Liposome loading

ABSTRACT

Provided herein is technology relating to incorporation of drugs into liposomes and particularly, but not exclusively, to methods for incorporating drugs into liposomes using a weak base and related compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/165,421, filed May 26, 2016, which claims priority to U.S.Provisional Patent Application No. 62/166,223, filed May 26, 2015 andU.S. Provisional Patent Application No. 62/291,225, filed Feb. 4, 2016,the contents of each of which are incorporated by reference in theirentireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No.1R43DA037887-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

Provided herein is technology relating to incorporation of bioactiveagents such as drugs into liposomes and particularly, but notexclusively, to methods and related compositions for incorporatingbioactive agents into liposomes using a weak base.

BACKGROUND

Controlled-release drug formulations can be produced by incorporatingdrugs into liposomes. These formulations have many advantages including,e.g., extending the duration of a drug's effect followingadministration. Important considerations related to these technologiesinclude the efficiency with which the drug is incorporated intoliposomes and the release profile of the drug from the liposomes.

Some existing methods for incorporating drugs into liposomes employpassive aqueous capture. At best, this method incorporates only 50% ofthe drug into the liposomes and release rates after administration arevery rapid. For example, previous data indicated that oxymorphoneincorporation into dehydration-rehydration vesicles comprising eggphosphatidylcholine and cholesterol was 50% efficient and subsequentanimal studies using these vesicles indicated that the release time wasapproximately 24 hours. In additional studies, incorporation of the druginto dehydration-rehydration vesicles comprisingdipalmitoylphosphatidylcholine and cholesterol was only 7% efficient,although release times were more favorable at approximately 72 hours. Inboth cases, release rates were most rapid at early time points,resulting in high initial plasma concentrations of the drug.

Accordingly, technologies that combine efficient incorporation of drugsinto liposomes with favorable release kinetics are needed.

SUMMARY

Gradient loading technologies provide an alternative to passive aqueouscapture. In general, gradient loading methods improve the efficiencywith which drugs are incorporated into liposomes. One illustrativeexample of this approach is the use of ammonium sulfate as a liposomeloading agent. In particular, liposomes are prepared in an ammoniumsulfate solution, which results in liposomes having ammonium sulfate inthe intraliposomal space. Ammonium sulfate is also present in theextraliposomal space (e.g., in the bulk phase). The unincorporatedammonium sulfate in the bulk phase is eliminated by dialysis or othertechnique. Then, the drug is added to the washed liposomes comprisingammonium sulfate in the intraliposomal space. As the systemequilibrates, the drug is loaded into the liposomes by the process ofweak base exchange. This approach is 99% efficient for a drug such asdoxorubicin, which precipitates as an insoluble sulfate after transportinto the liposomes. However, loading is less efficient for most drugsbecause most drug sulfate salts are highly soluble in aqueous solutions.For example, previous studies have indicated that liposomes are loadedwith opioid drugs (e.g., oxymorphone, hydromorphone, and buprenorphine)using the ammonium sulfate gradient technique at loading efficiencies ofapproximately 35%.

Despite such moderate loading, a benefit of ammonium sulfate loading isthe improved release profile of opioids loaded into liposomes. Inparticular, therapeutic concentrations of opioids in the blood aremaintained for 2 to 3 weeks after a single subcutaneous injection.However, as for passively loaded opioids, the early release rate israpid, which results in undesirably high plasma concentrations for thefirst few days following injection.

Accordingly, provided herein is technology that provides highlyefficient liposome loading with drugs and a reduced release rate (e.g.,a reduced early release rate).

For example, some embodiments of the technology provide a liposomecomposition for preparing liposomes loaded with a bioactive agent, thecomposition comprising liposomes, the liposomes comprising a loadingbase in the intraliposomal space, the loading base having a pKa that isless than the pKa of the bioactive agent. In some embodiments, thecompositions further comprise a loading medium in the extraliposomalspace, the loading medium having a pH that is greater than the pKa ofthe loading base. In particular embodiments, the pH of the loadingmedium is controlled by using a pH buffer in the loading medium (e.g.,in some embodiments the loading medium comprises a buffer; e.g., in someembodiments the extraliposomal space comprises a buffer). In someembodiments of the liposome composition for preparing liposomes, theconcentration of the loading base in the intraliposomal space is greaterthan the concentration of the loading base in the extraliposomal space.In some embodiments, the (log P−pKa) of the loading base is greater thanthe (log P−pKa) of the bioactive agent.

The liposome compositions for preparing liposomes loaded with abioactive agent are not limited in the loading base used. In someembodiments, the loading base is a weak base (e.g., the pKa of theloading base is less than 6, less than 5.5, less than 5, less than 4.5,less than 4, less than 3.5, less than 3, less than 2.5, or less than 2).In some embodiments, the loading base is a pyridine or pyridinederivative, e.g., a pyridinium, a 2-methoxy-pyridinium, a nicotinamideor a pyridazinium (e.g., a pyridinium ion, a 2-methoxy-pyridinium ion,or a pyridazinium ion; e.g., a pyridinium salt, a 2-methoxy-pyridiniumsalt, or a pyridazinium salt). In some embodiments, the log P of theloading base is less than 1.5, less than 1.0, less than 0.5, less than0.0, or less than −0.5. The technology is not limited in the lipids usedto prepare the liposomes. For example, in some embodiments the liposomescomprise phosphatidylcholine, e.g., a phosphatidylcholine selected fromthe group consisting of distearoylphosphatidylcholine, hydrogenated soyphosphatidylcholine, hydrogenated egg phosphatidylcholine,dipalmitoyl-phosphatidylcholine, dimyristoylphosphatidylcholine, anddielaidoylphosphatidylcholine. In some embodiments, the liposomescomprise a sphingomyelin; a neutral lipid; and/or an acidicphospholipid. Some embodiments of the technology are related toliposomes comprising a dipalmitoylphosphatidylcholine and, optionally,cholesterol.

In some embodiments, liposome compositions find use to prepare liposomesloaded with a bioactive agent. For example, some embodiments of thetechnology provide a composition comprising liposomes, a bioactiveagent, and a loading base, the loading base having a pKa that is lessthan a pKa of the bioactive agent. In an exemplary embodiment of thecompositions, the concentration of the bioactive agent in theintraliposomal space is greater than the concentration of the bioactiveagent in the extraliposomal space and the concentration of the loadingbase in the intraliposomal space is less than the concentration of theloading base in the extraliposomal space. In some embodiments, the (logP−pKa) of the loading base is greater than the (log P−pKa) of thebioactive agent.

The liposomes loaded with a bioactive agent are not limited in theloading base used for loading. In some embodiments, the loading base isa weak base (e.g., the pKa of the loading base is less than 6, less than5.5, less than 5, less than 4.5, less than 4, less than 3.5, less than3, less than 2.5, or less than 2). In some embodiments, the loading baseis a pyridine or pyridine derivative, e.g., a pyridinium, a2-methoxy-pyridinium, a nicotinamide or a pyridazinium (e.g., apyridinium ion, a 2-methoxy-pyridinium ion, or a pyridazinium ion; e.g.,a pyridinium salt, a 2-methoxy-pyridinium salt, or a pyridazinium salt).In some embodiments, the log P of the loading base is less than 1.5,less than 1.0, less than 0.5, less than 0.0, or less than −0.5.

Furthermore, the technology is not limited in the bioactive agent loadedinto the liposomes. For instance, some embodiments provide that thebioactive agent is an analgesic, e.g., an opioid. In some embodiments,the bioactive agent is hydromorphone, chloroquine, naltrexone, orbuprenorphine. In some embodiments, the bioactive agent is an antitumoragent, an anaesthetic, an analgesic, an antimicrobial agent, a hormone,an antiasthmatic agent, a cardiac glycoside, an antihypertensive, avaccine, an antiarrhythmic, an immunomodulator, a steroid, a monoclonalantibody, a neurotransmitter, a radionuclide, a radio contrast agent, anucleic acid, a protein, a herbicide, a pesticide, and/or suitablecombinations thereof.

Some embodiments are related to pharmaceutical compositions.Accordingly, in some embodiments the compositions provided comprise anexcipient and/or a pharmaceutically acceptable carrier.

The technology provides for the efficient loading of liposomes with abioactive agent. For example, in some embodiments the efficiency ofloading is from 80 to 100%, e.g., in some embodiments the amount ofbioactive agent in the intraliposomal space is greater thanapproximately 80%, greater than approximately 85%, greater thanapproximately 90%, or greater than approximately 95% of the total amountof bioactive agent in the composition.

Some embodiments provide a liposomal system comprising an aqueous mediumhaving dispersed therein liposomes encapsulating a loading base in theintraliposomal space; and a bioactive agent, wherein the loading basehas a pKa that is less than a pKa of the bioactive agent. In someembodiments of systems, the (log P−pKa) of the loading base is greaterthan the (log P−pKa) of the bioactive agent.

Associated embodiments of methods are provided. For example, in someembodiments the technology provides a method of preparing a compositionof liposomes for loading with a bioactive agent, the method comprisingpreparing liposomes in a solution comprising a salt of a loading base;and removing, substantially removing, essentially removing, effectivelyremoving, and/or decreasing the concentration of the salt of the loadingbase from the extraliposomal space (e.g., by sedimentation in acentrifuge, dialysis, dilution, gel chromatography, etc.) to produce acomposition of liposomes for loading with a bioactive agent, wherein theloading base has a pKa lower than the pKa of the bioactive agent. Someembodiments comprise a step of adding a loading medium to theextraliposomal space, the loading medium having a pH greater than thepKa of the loading base.

The methods for preparing liposome compositions for preparing liposomesloaded with a bioactive agent are not limited in the loading base usedin the provided methods. In some embodiments, the loading base is a weakbase (e.g., the pKa of the loading base is less than 6, less than 5.5,less than 5, less than 4.5, less than 4, less than 3.5, less than 3,less than 2.5, or less than 2). In some embodiments, the loading base isa pyridine or pyridine derivative, e.g., a pyridinium, a2-methoxy-pyridinium, or a pyridazinium (e.g., a pyridinium ion, a2-methoxy-pyridinium ion, a nicotinamide or a pyridazinium ion; e.g., apyridinium salt, a 2-methoxy-pyridinium salt, or a pyridazinium salt).In some embodiments, the log P of the loading base is less than 1.5,less than 1.0, less than 0.5, less than 0.0, or less than −0.5.

Further embodiments of methods are related to loading liposomes with abioactive agent. For example, some embodiments provide methods forpreparing liposomes encapsulating a bioactive agent, the methodcomprising providing a composition comprising liposomes, the liposomescomprising a loading base in an intraliposomal space; and adding abioactive agent to the composition, wherein the loading base has a pKathat is lower than the pKa of the bioactive agent. Some embodimentscomprise a step of providing a loading medium in the extraliposomalspace comprising a pH that is greater than the pKa of the bioactiveagent.

The methods for loading liposomes with a bioactive agent are not limitedin the loading base used in the liposome loading methods. In someembodiments, the loading base is a weak base (e.g., the pKa of theloading base is less than 6, less than 5.5, less than 5, less than 4.5,less than 4, less than 3.5, less than 3, less than 2.5, or less than 2).In some embodiments, the loading base is a pyridine or pyridinederivative, e.g., a pyridinium, a 2-methoxy-pyridinium, or apyridazinium (e.g., a pyridinium ion, a 2-methoxy-pyridinium ion, anicotinamide or a pyridazinium ion; e.g., a pyridinium salt, a2-methoxy-pyridinium salt, or a pyridazinium salt). In some embodiments,the log P of the loading base is less than 1.5, less than 1.0, less than0.5, less than 0.0, or less than −0.5.

Some embodiments provide a liposome composition for preparing liposomesloaded with a bioactive agent, the composition comprising liposomes, theliposomes comprising a diprotic acid or a salt of a diprotic acid,wherein the first pKa and the second pKa of the diprotic acid are bothless than zero. For example, in some embodiments the diprotic acid is asulfonate, e.g., a disulfonate such as, e.g., methanedisulfonic acid;1,2-ethanedisulfonic acid (edisylate); or 1,3-propanedisulfonic acid(eprodisate). Related embodiments provide a liposomal system comprisingan aqueous medium having dispersed therein liposomes encapsulating adiprotic acid or a salt of a diprotic acid, wherein the first pKa andthe second pKa of the diprotic acid are both less than zero; and abioactive agent. In some embodiments, the diprotic acid is a sulfonate,e.g., a disulfonate such as, e.g., methanedisulfonic acid;1,2-ethanedisulfonic acid (edisylate); or 1,3-propanedisulfonic acid(eprodisate). Embodiments of methods for producing a composition ofliposomes for loading with a bioactive agent comprise the steps ofpreparing liposomes in a solution comprising a diprotic acid or a saltof a diprotic acid, wherein the first pKa and the second pKa of thediprotic acid are both less than zero; and removing the diprotic acid ora salt of a diprotic acid from the extraliposomal space to produce acomposition of liposomes for loading with a bioactive agent. In someembodiments of the methods, the diprotic acid is a sulfonate (e.g.,disulfonate), e.g., methanedisulfonic acid; 1,2-ethanedisulfonic acid(edisylate); or 1,3-propanedisulfonic acid (eprodisate). Relatedembodiments of methods for preparing liposomes comprising a bioactiveagent comprise providing a composition comprising liposomes, theliposomes comprising a diprotic acid or a salt of a diprotic acid,wherein the first pKa and the second pKa of the diprotic acid are bothless than zero; and adding a bioactive agent to the composition. In someembodiments, the diprotic acid is a sulfonate, e.g., a disulfonate suchas, e.g., methanedisulfonic acid; 1,2-ethanedisulfonic acid (edisylate);or 1,3-propanedisulfonic acid (eprodisate).

Also provided are methods for producing liposomes by dissolving lipidsin a solvent to produce a lipid solution; adding a weak base salt (e.g.,a sulfate, an eprodisate, or an edisylate) to the lipid solution toproduce liposomes. In some embodiments, the lipids are dry powders. Insome embodiments, the solvent is an alcohol. In some embodiments, thesolvent is miscible with aqueous solutions and/or the solvent hassignificant aqueous solubility, e.g., the alcohol is sufficientlysoluble in water to form a single phase at the desired alcohol to waterratio, e.g., an alcohol that has an aqueous solubility of at least 50g/L (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, or 150 g/L aqueous solubility) or, morepreferably, an aqueous solubility of more than 150 g/L (e.g., more than150, 160, 170, 180, 190, 200, 250, 300 g/L, or more g/L, or completelymiscible at any ratio).

For example, in some embodiments the solvent is an alcohol such as,e.g., 2-propanol, t-butanol, methanol, ethanol, 1-propanol, 2-butanol,isobutanol, 1-butanol, and/or 1-pentanol and isomers of 1-pentanol.However, the technology is not limited to these solvents. Any solventthat provides for the production of liposomes according to thetechnology provided herein.

For example, data collected during the development of embodiments of thetechnology indicate that efficient liposome loading is provided bymethods that comprise use of an alcohol that is sufficiently nonpolar tosolvate the lipids used to prepare the liposomes and partitionsignificantly into the hydrophobic (e.g., oil) phase while also beingsufficiently polar to allow the alcohol to be washed away and/or dilutedonce the liposomes are prepared. Accordingly, embodiments comprise useof an alcohol that has a Log P value that is between approximately −0.2to 1.0 and preferably between approximately 0.1 to 0.8. Alternatively,the alcohol may be chosen based on the number of carbons in the alcohol.For example, in some embodiments the alcohol used to prepare liposomeshas 1 to 6 carbon atoms (e.g., 1, 2, 4, 5, 6, or 6 carbon atoms). Insome particular embodiments, the alcohol has 3 or 4 carbon atoms.

In some embodiments, the dissolving comprises warming the lipids andsolvent to 40 to 85° C. or 40° C. to 70° C. In some embodiments, themethod comprises warming the weak base salt to 40 to 85° C. or 40° C. to70° C. prior to adding the weak base salt to the lipid solution. Relatedembodiments comprise an additional step of cooling the liposomes to atemperature that is below the phase transition temperature of the lipidsand, in some embodiments, the methods comprise a further step ofdiluting the liposomes in an aqueous solution. In some embodiments themethods comprise washing the liposomes to remove weak base salt from theextraliposomal space. In some embodiments, the solvent is methanol,ethanol, 1-propanol, 2-propanol, 2-butanol, 1-butanol or t-butanol.

In particular embodiments, the weak base salt is added to the lipidsolution in a plurality of volumes (e.g., two or more volumes that areadded separately to the lipid solution). For example, in someembodiments adding the weak base salt to the lipid solution comprisesadding a first volume of the weak base salt to the lipid solutionfollowed by adding a second volume of the weak base salt to the lipidsolution. In some embodiments, the ratio of the first volume to thesecond volume is 5:1 to 1:5 (e.g., 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1,2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5).

Embodiments of the method find use in encapsulating a bioactive agent inthe intraliposomal space. Accordingly, the technology providesembodiments of methods comprising providing a composition of liposomesprepared by a method as described above; and adding a bioactive agent tothe composition of liposomes.

Associated methods relate to a method of treating a subject in need ofpain reduction, the method comprising administering to the subject acomposition as described herein. In some embodiments, methods oftreating a subject further comprise assessing the subject's pain.

Additional embodiments will be apparent to persons skilled in therelevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presenttechnology will become better understood with regard to the followingdrawings:

FIG. 1 is a plot showing the theoretical loading curves for loading ofhydromorphone into liposomes containing 20 μmol ammonium or pyridinium.

FIG. 2 is a plot showing leakage of chloroquine from 0.5 M pyridiniumsulfate-containing, chloroquine-loaded liposomes.

FIG. 3 is a plot showing leakage of pyridine from 0.5 M pyridiniumsulfate-containing, chloroquine-loaded liposomes.

FIG. 4 is a plot showing leakage of chloroquine from 1 M pyridiniumchloride-containing, chloroquine-loaded liposomes.

FIG. 5 is a plot showing leakage of pyridine from 1 M pyridiniumchloride-containing, chloroquine-loaded liposomes.

FIG. 6 is a plot showing leakage of chloroquine from 0.133 M ammoniumsulfate-containing, chloroquine-loaded liposomes.

FIG. 7 is a plot showing leakage of hydromorphone from 0.5 M pyridiniumsulfate-containing, hydromorphone-loaded liposomes.

FIG. 8 is a plot showing leakage of naltrexone from 1.5 M pyridiniumsulfate-containing, naltrexone-loaded liposomes.

FIG. 9 is a plot showing leakage of buprenorphine from liposomes whenthe buprenorphine is loaded using pyridinium sulfate (diamonds),2-methoxypyridinium sulfate (triangles), 2-methoxypyridinium triflate(squares), and pyridazinium triflate (circles).

FIG. 10 is a plot showing leakage of chloroquine from liposomes loadedwith adenine triflate (squares), aniline triflate (circles), or pyridinesulfate (diamonds).

FIG. 11 is a plot showing leakage of doxycycline from liposomes loadedwith 2-methoxypyridinium sulfate.

FIG. 12 is a plot showing leakage of buprenorphine from liposomes loadedwith hydrochloric acid (diamonds) and eprodisic acid (squares).

FIG. 13 is a plot showing release of chloroquine from DPPC:cholesterolliposomes loaded using 0.5 M pyridinium eprodisate (triangles) or 0.5 M2-methoxypyridinium eprodisate (circles) compared to pyridinium sulfate(squares) and 2-methoxypyridinium sulfate (diamonds).

FIG. 14 is a plot showing serum buprenorphine concentrations following asingle 2 mg/Kg injection of buprenorphine in DPPC:cholesterol (diamonds)or DSPC:cholesterol (triangles) liposomes loaded with 0.24 M2-methoxypyridinium sulfate.

FIG. 15 is a plot showing serum buprenorphine concentrations following asingle 2 mg/Kg injection of buprenorphine in DPPC:cholesterol (diamonds)or DSPC:cholesterol (squares) liposomes loaded with 0.24 M2-methoxypyridinium eprodisate.

FIG. 16 is a plot showing the leakage of naltrexone from liposomesprepared with pyridinium salts of sulfate (filled triangles), eprodisate(filled squares), methane sulfonate (“mesylate”, open triangles),benzenesulfonate (open circles), chloride (open diamonds), and nitrate(open squares).

It is to be understood that the figures are not necessarily drawn toscale, nor are the objects in the figures necessarily drawn to scale inrelationship to one another. The figures are depictions that areintended to bring clarity and understanding to various embodiments ofapparatuses, systems, and methods disclosed herein. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or like parts. Moreover, it should be appreciated that thedrawings are not intended to limit the scope of the present teachings inany way.

DETAILED DESCRIPTION

Provided herein is a technology related to using weak bases as theloading base to load drugs efficiently into liposomes. In particular,experiments were conducted indicating that drugs such as chloroquine,hydromorphone, naltrexone, and buprenorphine are loaded into liposomeswith high efficiency using a weak base such as pyridine and relatedmolecules (e.g., 2-methoxypridine, pyridazine, etc.) or nicotinamide.The data collected indicated that the technology provides a high loadingefficiency and the in vitro leakage of the drug from the liposomes inthe early phase of leakage (0-48 hrs) was very low. In some embodiments,the weak loading bases act as sacrificial leakage agents that are leakedfrom the liposomes rather than the drug.

In this detailed description of the various embodiments, for purposes ofexplanation, numerous specific details are set forth to provide athorough understanding of the embodiments disclosed. One skilled in theart will appreciate, however, that these various embodiments may bepracticed with or without these specific details. In other instances,structures and devices are shown in block diagram form. Furthermore, oneskilled in the art can readily appreciate that the specific sequences inwhich methods are presented and performed are illustrative and it iscontemplated that the sequences can be varied and still remain withinthe spirit and scope of the various embodiments disclosed herein. Thesection headings used herein are for organizational purposes only andare not to be construed as limiting the described subject matter in anyway.

All literature and similar materials cited in this application,including but not limited to, patents, patent applications, articles,books, treatises, and internet web pages are expressly incorporated byreference in their entirety for any purpose. Unless defined otherwise,all technical and scientific terms used herein have the same meaning asis commonly understood by one of ordinary skill in the art to which thevarious embodiments described herein belongs. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control.

Definitions

To facilitate an understanding of the present technology, a number ofterms and phrases are defined below. Additional definitions are setforth throughout the detailed description.

Throughout the specification and claims, the following terms take themeanings explicitly associated herein, unless the context clearlydictates otherwise. The phrase “in one embodiment” as used herein doesnot necessarily refer to the same embodiment, though it may.Furthermore, the phrase “in another embodiment” as used herein does notnecessarily refer to a different embodiment, although it may. Thus, asdescribed below, various embodiments of the technology may be readilycombined, without departing from the scope or spirit of the technology.

In addition, as used herein, the term “or” is an inclusive “or” operatorand is equivalent to the term “and/or” unless the context clearlydictates otherwise. The term “based on” is not exclusive and allows forbeing based on additional factors not described, unless the contextclearly dictates otherwise. In addition, throughout the specification,the meaning of “a”, “an”, and “the” include plural references. Themeaning of “in” includes “in” and “on.”

The term “lipid” refers to any suitable material resulting in a bilayersuch that the hydrophobic portion of the lipid material orients towardthe bilayer interior while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofphosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other likegroups. Hydrophobicity could be conferred by the inclusion of groupsthat include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups and such groups substituted byone or more aromatic, cycloaliphatic or heterocyclic group(s).

Amphipathic lipids often find use as the primary lipid vesiclestructural element. Examples of amphipathic compounds arephosphoglycerides and sphingolipids, representative examples of whichinclude phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoyl-phosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid and glycosphingolipid families are also within thegroup designated as lipid. Additionally, the amphipathic lipidsdescribed above may be mixed with other lipids including triacyglycerolsand sterols.

“Phospholipid” refers to any one phospholipid or combination ofphospholipids capable of forming liposomes. Phosphatidylcholines (PC),including those obtained from egg, soy beans, or other plant sources orthose that are partially or wholly synthetic, or of variable lipid chainlength and unsaturation find use in embodiments of the presenttechnology. Synthetic, semisynthetic, and natural productphosphatidylcholines including, but not limited to,distearoylphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), soy phosphatidylcholine (soy PC), eggphosphatidylcholine (egg PC), hydrogenated egg phosphatidylcholine(HEPC), dipalmitoylphosphatidylcholine (DPPC), anddimyristoylphosphatidylcholine (DMPC) are suitable phosphatidylcholinesfor use in this technology. All of these phospholipids are commerciallyavailable.

Further, phosphatidylglycerols (PG) and phosphatic acid (PA) are alsosuitable phospholipids for use in the present technology and include,but are not limited to, dimyristoylphosphatidylglycerol (DMPG),dilaurylphosphatidylglycerol (DLPG), dipalmitoylphosphatidylglycerol(DPPG), distearoylphosphatidylglycerol (DSPG) dimyristoylphosphatidicacid (DMPA), distearoylphosphatidic acid (DSPA), dilaurylphosphatidicacid (DLPA), and dipalmitoylphosphatidic acid (DPPA). Other suitablephospholipids include phosphatidylethanolamines, phosphatidylinositols,and phosphatidic acids containing lauric, myristic, stearoyl, andpalmitic acid chains. Further, incorporation of polyethylene glycol(PEG) containing phospholipids is also contemplated by the presenttechnology. It is contemplated by this technology to include cholesteroloptionally in the liposomal formulation. Cholesterol is known to improveliposome stability and prevent loss of phospholipid to lipoproteins invivo.

“Unilamellar liposomes”, also referred to as “single lamellar vesicles,”are spherical vesicles that include one lipid bilayer membrane thatdefines a single closed aqueous compartment. The bilayer membraneincludes two layers (or “leaflets”) of lipids; an inner layer and anouter layer. The outer layer of the lipid molecules is oriented with thehydrophilic head portions toward the external aqueous environment andthe hydrophobic tails pointed downward toward the interior of theliposome. The inner layer of the lipid lay directly beneath the outerlayer with the lipids oriented with the heads facing the aqueousinterior of the liposome and the tails oriented toward the tails of theouter layer of lipid.

“Multilamellar liposomes” also referred to as “multilamellar vesicles”or “multiple lamellar vesicles,” include more than one lipid bilayermembrane, which membranes define more than one closed aqueouscompartment. The membranes are concentrically arranged so that thedifferent membranes are separated by aqueous compartments, much like anonion.

The terms “bioactive agent” and “pharmaceutical agent” (e.g., a “drug”)are used interchangeably and include but are not limited to, anantibiotic, an analgesic, an anesthetic, an antiacne agent, anantibiotic, an antibacterial, an anticancer agent, an anticholinergic,an anticoagulant, an antidyskinetic, an antiemetic, an antifibrotic, anantifungal, an antiglaucoma agent, an anti-inflammatory, anantineoplastic, an antiosteoporotic, an antipagetic, an anti-Parkinson'sagent, an antisporatic, an antipyretic, an antiseptic, anantithrombotic, an antiviral, an antimalarial, an antiparasitic, acalcium regulator, a keratolytic, and/or a sclerosing agent.

The terms “encapsulation” and “entrapped,” as used herein, refer to theincorporation or association of a biologically active (e.g., apharmaceutical agent) in or with a liposome. The pharmaceutical agentmay be associated with the lipid bilayer or present in the aqueousinterior (“intraliposomal space”) of the liposome, or both. In oneembodiment, a portion of the encapsulated pharmaceutical agent takes theform of a precipitated salt in the interior of the liposome. Thepharmaceutical agent may also self-precipitate in the interior of theliposome.

As used herein, a “liposome loading agent” refers to a substance (e.g.,chemical, molecule, etc.) that promotes the movement of anothersubstance (e.g., a drug) into the intraliposomal space (e.g., the lumen)of a liposome. The liposome loading agent may preferably be a counterionor counterion excipient that can initiate or facilitate drug loading andmay also initiate or facilitate precipitation of the pharmaceuticalagent in the aqueous interior of the liposome. Examples of liposomeloading agents include, but are not limited to, weak bases and saltsthereof as well as the acid, sodium or ammonium forms of monovalentanions such as chloride, acetate, lactobionate and formate; divalentanions such as aspartate, succinate and sulfate; and trivalent ions suchas citrate and phosphate.

As used herein, the term “log P” refers to the logarithm of thepartition coefficient (P) describing the ratio of concentrations of acompound in a mixture of two immiscible phases at equilibrium. Inparticular, the log P as used herein refers to the distribution betweentwo reference phases, e.g., water and a non-aqueous-miscible liquid,e.g., an organic solvent, e.g., n-octanol. In some embodiments, log Pvalues are theoretically calculated, e.g., using a group additivityapproach or, in some embodiments, a method including factors such asdipole moment, molecular size, molecular shape, etc. See, e.g.,Viswanadhan, et al (1989, J. Chem. Inf. Comput. Sci. 29(3): 163; Suzukiand Kudo (1990), J. Comput. Aided Mol. Des. 4(2): 155-98, eachincorporated herein by reference. Thus, the partition coefficient is ameasure of how hydrophilic or hydrophobic a chemical substance is.Accordingly, partition coefficients are useful in estimating thedistribution of drugs within the body.

As used herein, the “loading efficiency” refers to the amount of asubstance that is incorporated into a liposome (e.g., in theintraliposomal space) by a liposome loading process relative to thetotal amount of the substance added. Generally, a liposome preparationis prepared for loading with a substance such as a drug; then, a knowninitial amount of the substance (e.g., drug) is added to thepreparation. The substance (e.g., drug) is initially in theextraliposomal space and some amount of the substance (e.g., drug) movesinto and becomes entrapped in the intraliposomal space. The ratio (e.g.,expressed as a percentage, fraction, ratio, etc.) of entrapped substance(e.g., drug) relative to the known initial amount of the substance(e.g., drug) is a measure of the loading efficiency.

As used herein, “treat” or “treating” refers to: (i) preventing apathologic condition (e.g., breast cancer; sepsis) from occurring (e.g.prophylaxis) or preventing symptoms related to the same; (ii) inhibitingthe pathologic condition or arresting its development or inhibiting orarresting symptoms related to the same; or (iii) relieving thepathologic condition or relieving symptoms related to the same.

As used herein, the terms “subject” and “patient” refer to any animal,such as a mammal like a dog, cat, bird, livestock, and preferably ahuman.

As used herein, the term “effective amount” refers to the amount of acomposition sufficient to effect beneficial or desired results. Aneffective amount can be administered in one or more administrations,applications, or dosages and is not intended to be limited to aparticular formulation or administration route.

As used herein, the term “administration” refers to the act of giving adrug, prodrug, or other agent, or therapeutic treatment to a subject.Exemplary routes of administration to the human body can be through theeyes (ophthalmic), mouth (oral), skin (transdermal, topical), nose(nasal), lungs (inhalant), oral mucosa (buccal), ear, by injection(e.g., intravenously, subcutaneously, intratumorally, intraperitoneally,etc.), and the like.

As used herein, the term “pharmaceutical composition” refers to thecombination of a biological agent with a carrier, inert or active,making the composition especially suitable for therapeutic use.

The terms “pharmaceutically acceptable” or “pharmacologicallyacceptable”, as used herein, refer to compositions that do notsubstantially produce adverse reactions, e.g., toxic, allergic, orimmunological reactions, when administered to a subject.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a disease or disorder throughintroducing in any way a therapeutic composition of the presenttechnology into or onto the body of a subject. “Treatment” refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) the targetedpathologic condition or disorder. Those in need of treatment includethose already with the disorder as well as those prone to have thedisorder or those in whom the disorder is to be prevented.

As used herein, “therapeutically effective dose” refers to an amount ofa therapeutic agent sufficient to bring about a beneficial or desiredclinical effect. The dose can be administered in one or moreadministrations. However, the precise determination of what would beconsidered an effective dose may be based on factors individual to eachpatient, including, but not limited to, the patient's age, size, type orextent of disease, stage of the disease, route of administration, thetype or extent of supplemental therapy used, ongoing disease process,and type of treatment desired (e.g., aggressive versus conventionaltreatment).

Description

The technology provided herein relates to the use of weak bases as aloading base to load drugs efficiently into liposomes. Exemplary weakbases include but are not limited to pyridine, 2-methoxypyridine,pyridazine, and nicotinamide.

Liposomes

Liposomes, or lipid vesicles, are used for drug delivery to improve thetherapeutic activity and increase the safety of a number of differentpharmaceutical agents. Liposomal carrier systems (e.g., vesicles) aremicroscopic spheres of one or more lipid bilayers arranged around anaqueous core. The vesicles have been shown to be suitable as carriersfor both hydrophilic and hydrophobic therapeutic agents owing to theirunique combination of lipophilic and hydrophilic portions.

Liposomes are completely closed lipid bilayer membranes containing anentrapped volume. The bilayer membrane separates this surrounded volume(the “intraliposomal space” or “lumen”) from the bulk phase (the“extraliposomal space”). Liposomes may be unilamellar vesicles(possessing a single membrane bilayer) or multilameller vesicles(onion-like structures characterized by multiple membrane bilayers, eachseparated from the next by an aqueous layer). Liposomes may take otherforms as well, e.g., multivesicular liposomes (MVL), which are lipidvesicles with multiple internal aqueous chambers formed bynon-concentric layers and having internal membranes distributed as anetwork throughout the MVL.

In these various forms, the bilayer is composed of two lipid monolayershaving a hydrophobic “tail” region and a hydrophilic “head” region. Thestructure of the membrane bilayer is such that the hydrophobic(nonpolar) “tails” of the lipid monolayers orient toward the center ofthe bilayer while the hydrophilic “heads” orient towards the aqueousphase.

Liposome Formation

In a conventional liposome preparation such as that of Bangham et al.(J. Mol. Biol., 1965, 13: 238-252), phospholipids were suspended in anorganic solvent that was then evaporated to dryness to leave aphospholipid film on the reaction vessel. Next, an appropriate amount ofaqueous phase was added, the mixture was allowed to “swell”, and theresulting MLVs were dispersed by mechanical means to producemultilamellar vesicles. This preparation provided the basis for thedevelopment of the small sonicated unilamellar vesicles described byPapahadjopoulos et al. (Biochim. Biophys, Acta., 1967, 135: 624-638) andmultilamellar vesicles.

Subsequently, techniques for producing large unilamellar vesicles (LUVs)such as reverse phase evaporation, infusion procedures, and detergentdilution were used to produce liposomes. A review of these and othermethods for producing liposomes may be found in the text Liposomes, MarcOstro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1. See alsoSzoka Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467). Oneparticular method for forming LUVs is described in Cullis et al., PCTPublication No. 87/00238, Jan. 16, 1986, entitled “Extrusion Techniquefor Producing Unilamellar Vesicles”.

In some embodiments, liposomes that are used in the present technologyare formed from standard vesicle-forming lipids, which generally includeneutral and negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of, e.g., liposome size and stability of the liposomes inthe bloodstream.

Various types of lipids are used to produce liposomes. For example,amphipathic lipids that find use are zwitterionic, acidic, or cationiclipids. Examples of zwitterionic amphipathic lipids arephosphatidylcholines, phosphatidyl-ethanolamines, sphingomyelins, etc.Examples of acidic amphipathic lipids are phosphatidylglycerols,phosphatidylserines, phosphatidylinositols, phosphatidic acids, etc.Examples of cationic amphipathic lipids are diacyl trimethylammoniumpropanes, diacyl dimethylammonium propanes, stearylamine, etc. Examplesof neutral lipids include diglycerides, such as diolein, dipalmitolein,and mixed caprylin-caprin; triglycerides, such as triolein,tripalmitolein, trilinolein, tricaprylin, and trilaurin; andcombinations thereof. Additionally, cholesterol or plant sterols areused in some embodiments, e.g., to make multivesicular liposomes.

In some embodiments, the major lipid component in the liposomes isphosphatidylcholine. Phosphatidylcholines having a variety of acyl chaingroups of varying chain length and degree of saturation are available ormay be isolated or synthesized by well-known techniques. In general,less saturated phosphatidylcholines are more easily sized, particularlywhen the liposomes must be sized below approximately 0.3 microns, e.g.,for purposes of filter sterilization. In some embodiments,phosphatidylcholines containing saturated fatty acids with carbon chainlengths in the range of approximately C₁₄ to C₂₂ are preferred.Phosphatidylcholines with monounsaturated or diunsaturated fatty acidsand mixtures of saturated and unsaturated fatty acids are used in someembodiments. Other suitable lipids include phosphonolipids in which thefatty acids are linked to glycerol via ether linkages rather than esterlinkages (e.g., as found in some members of the Archaea). Liposomesuseful in the present technology may also be composed of sphingomyelinor phospholipids with head groups other than choline, such asethanolamine, serine, glycerol, and inositol. In some embodiments,liposomes include a sterol, preferably cholesterol, at molar ratios offrom 0.1 to 1.0 of the cholesterol to the phospholipid). In someembodiments, the liposome compositions aredistearoylphosphatidylcholine/cholesterol,dipalmitoylphosphatidylcholine/cholesterol, orsphingomyelin/cholesterol. Methods used in sizing and filter-sterilizingliposomes are provided below.

A variety of methods are available for preparing liposomes as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871; 4,501,728; and 4,837,028; the text Liposomes, MarcJ. Ostro, ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, and Hope,et al., Chem. Phys. Lip. 40:89 (1986), each of which is incorporatedherein by reference. One exemplary method produces multilamellarvesicles of heterogeneous sizes. In this method, the vesicle-forminglipids are dissolved in a suitable organic solvent or solvent system anddried under vacuum or an inert gas to form a thin lipid film.Alternatively, the lipids may be dissolved in a suitable solvent, suchas tertiary butanol, and then lyophilized to form a more homogeneouslipid mixture that is in a more easily hydrated, microporous,powder-like form. This film or powder is covered with an aqueoussolution (e.g., in some embodiments, an aqueous buffered solution) andallowed to hydrate, typically over a 15-60 minute period with agitation.The size distribution of the resulting multilamellar vesicles can beshifted toward smaller sizes by hydrating the lipids under more vigorousagitation conditions or by adding solubilizing detergents such asdeoxycholate.

Many different types of organic solvents such as ethers, hydrocarbons,halogenated hydrocarbons, and/or freons are used in some embodiments asthe solvent in the lipid component. For example, diethyl ether,isopropyl ether, and other ethers; chloroform; tetrahydrofuran;halogenated ethers; esters, and combinations thereof find use in thepresent technology.

Several techniques are available for sizing liposomes to a desired size.One sizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles less than approximately 0.05 microns in size.Homogenization is another method that relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenapproximately 0.1 and 0.5 microns, are observed. In both methods, theparticle size distribution can be monitored by conventional laser-beamparticle size discrimination.

In some embodiments, extrusion of liposomes through a small-porepolycarbonate membrane or an asymmetric ceramic membrane provides aneffective method for reducing liposome sizes to a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposome sizedistribution is achieved. The liposomes may be extruded throughsuccessively smaller-pore membranes to achieve a gradual reduction inliposome size. In some embodiments comprising use of extrusion methods,liposomes find use that have a size of from approximately 0.05 micronsto approximately 0.15 microns. In some embodiments, liposomes are notextruded. For example, in some embodiments the liposomes areapproximately 1 micron to 10 microns in diameter. While manytechnologies and sizes for liposomes are discussed herein, thetechnology is not dependent on the size of the liposomes; accordingly,there is no size preference for the liposome loading technology per se.

In some embodiments, liposomes are prepared, for example, by weighingout a quantity of a phosphatidylcholine (optionally cholesterol and/oroptionally a phosphatidylglycerol) and dissolving them in an organicsolvent, e.g., chloroform and methanol in a 1:1 mixture (v/v) oralternatively in neat chloroform. The solution is evaporated to form asolid lipid phase such as a film or a powder, for example, with a rotaryevaporator, spray dryer, or other method. The film or powder is thenhydrated with an aqueous solution optionally containing an excipient andhaving a pH range from approximately 2.0 to approximately 7.4 to form aliposome dispersion. The lipid film or powder dispersed in the aqueoussolution is heated to a temperature from approximately 25° C. toapproximately 70° C. depending on the phospholipids used.

Multilamellar liposomes are formed, e.g., by agitation of thedispersion, preferably through the use of a thin-film evaporatorapparatus such as is described in U.S. Pat. No. 4,935,171 or throughshaking or vortex mixing. Unilamellar vesicles are formed by theapplication of a shearing force to an aqueous dispersion of the lipidsolid phase, e.g., by sonication or the use of a microfluidizingapparatus such as a homogenizer or a French press. Shearing force canalso be applied using injection, freezing and thawing, dialyzing away adetergent solution from lipids, or other known methods used to prepareliposomes. The size of the liposomes can be controlled using a varietyof known techniques including controlling the duration of shearingforce. In some embodiments, a homogenizing apparatus is employed toproduce unilamellar vesicles having diameters of less than 200nanometers at a pressure of 3,000 to 14,000 psi (e.g., 10,000 to 14,000psi) and a temperature that is approximately at the aggregate transitiontemperature of the lipids. In some exemplary embodiments, liposomes areprepared as described below in the Methods section of the includedExamples.

According to some embodiments, liposomes are produced by combininglipids in chloroform, removing solvent to create a component mixture,suspending the lipid in a suitable liquid (e.g., an alcohol such as,e.g., t-butanol), and lyophilizing the suspension. Then, according tosome embodiments for loading liposomes with a bioactive agent, themicroporous lipid mass is subsequently hydrated using a weak base salt.In addition, provided herein are embodiments of the technology thateliminate the first steps (e.g., dissolving lipids in chloroform andremoving the solvent) commonly used for the preparation of liposomes.For example, some embodiments comprise a step of dissolving lipids in asuitable liquid (e.g., an alcohol such as, e.g., t-butanol) directlywithout a preceding step of mixing the lipid components in chloroform.In some embodiments, dissolving lipids in a suitable liquid (e.g., analcohol such as, e.g., t-butanol) is associated with heating the liquidto facilitate dissolving the lipid in the liquid. In some embodiments,the heating comprises providing an amount of heat to the liquid (e.g., aliquid comprising the lipid) that raises the temperature of the liquidsufficiently to dissolve the lipids therein (e.g. raising thetemperature by 1 to 60 degrees (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 degrees Celsius).

Additional embodiments of liposome preparation methods provided hereineliminate the lyophilizing step. For example, some embodiments prepareliposomes directly from lipids provided as dry powders. In particular,some embodiments of the methods described herein comprise providinglipids (e.g., phospholipid, cholesterol, etc.) as powders, dissolvingthe lipids in a suitable liquid (e.g., an alcohol such as, e.g., 1- or2-propanol), and adding a base salt to the lipid solution to produceliposomes. In some embodiments, dissolving lipids in a suitable liquid(e.g., an alcohol such as, e.g., 1- or 2-propanol) is associated withheating the liquid to facilitate dissolving the lipid in the liquid. Insome embodiments, the heating comprises providing an amount of heat tothe liquid (e.g., a liquid comprising the lipid) that raises thetemperature of the liquid sufficiently to dissolve the lipids therein(e.g. raising the temperature by 1 to 20 degrees (e.g., 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or60 degrees Celsius). During the development of embodiments of thetechnology provided herein, experiments were conducted to evaluateproducing liposomes directly from lipids (e.g., powdered lipids). See,e.g., Examples 10-14. For example, data collected from these experimentsindicated that producing liposomes directly from lipids (e.g., powderedlipids) is efficient—the methods incorporated approximately 25% of thebase salt into the liposomes and the methods form liposomes that arecomparable in their properties to those formed by present methodscomprising hydrating lyophilized lipid mixtures. Further, the liposomescan be readily washed by sedimentation at low g forces. In someembodiments, the liposomes are formed from dipalmitoylphosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC),and/or a hydrogenated soybean phospholipid comprising a mixture ofstearoyl and palmitoyl phosphatidyl choline.

Accordingly, in some embodiments, the present invention provides ofproducing liposomes, the method comprising: a) dissolving lipids in asolvent to produce a lipid solution; b) adding an aqueous solution tothe lipid solution to produce liposomes. In some embodiments, thesolvent is miscible with aqueous solutions or is a solvent that hassignificant aqueous solubility. In some embodiments, the solvent is analcohol. In some embodiments, the solvent is selected from the groupconsisting of methanol, ethanol, 1-propanol, 2-propanol, 2-butanol,1-butanol and t-butanol. In some embodiments, the solvent is selectedfrom the group consisting of a C3 and a C4 alcohol. In some embodiments,the C3 alcohol is 1-propanol. In some embodiments, the C4 alcohol is2-butanol.

In some embodiments, the lipids comprise phospholipids. In someembodiments, the phospholipids and the solvent are combined at aconcentration of about 400 mg to 800 mg phospholipids per 1 ml ofsolvent. In some embodiments, the phospholipids and the solvent arecombined at a concentration of about 500 mg to 700 mg phospholipids per1 ml of solvent. In some embodiments, the phospholipids and the solventare combined at a concentration of about 550 mg to 650 mg phospholipidsper 1 ml of solvent.

In some embodiments, the lipids further comprise cholesterol. In someembodiments, the cholesterol and the solvent are combined at aconcentration of about 50 mg to 250 mg cholesterol per 1 ml of solvent.In some embodiments, the cholesterol and the solvent are combined at aconcentration of about 100 mg to 200 mg cholesterol per 1 ml of solvent.In some embodiments, the cholesterol and the solvent are combined at aconcentration of about 125 mg to 175 mg cholesterol per 1 ml of solvent.

In some embodiments, the dissolving step comprises warming the lipidsand solvent to 40 to 85° C. or 40 to 70° C. In some embodiments, themethods further comprise warming the aqueous solution to 40 to 85° C. or40 to 70° C. prior to addition to the lipid solution. In someembodiments, the lipids and solvent are dissolved in a vessel to providea dissolved lipid composition and the aqueous solution is injected intothe vessel containing the dissolved lipid composition. In someembodiments, the methods further comprise cooling the liposomes to atemperature that is below the phase transition temperature of thelipids.

In some embodiments, the aqueous solution comprises a liposome loadingagent as described elsewhere herein. In some embodiments, the aqueoussolution is selected from the group consisting of an acidic solution anda basic solution. In some embodiments, the aqueous solution comprisesliposome loading agent selected from the group consisting of counterions and salts thereof. In some embodiments, the aqueous solutioncomprises a weak base salt selected from the group consisting of asulfate, an eprodisate, and an edisylate. In some embodiments, addingthe weak base salt to the lipid solution comprises adding a first volumeof the weak base salt to the lipid solution followed by adding a secondvolume of the weak base salt to the lipid solution. In some embodiments,the ratio of the first volume to the second volume is 5:1 to 1:5.

In some embodiments, the aqueous solution comprises an encapsulant,e.g., a compound or molecule that is designated to be encapsulated intothe intraliposomal space. Examples of encapsulants, and particularlybioactive agents, are described in detail herein. In some embodiments,the encapsulant is selected from the group consisting of a chemicalbioactive agent and a biologic bioactive agent. In some embodiments, thechemical bioactive agent is an analgesic.

In some embodiments, the methods further comprise diluting the liposomesin an aqueous solution. In some embodiments, the methods furthercomprise washing the liposomes to remove liposome loading agents orencapsulants, if utilized, from the extraliposomal space.

In some embodiments, the present invention provides methods forpreparing liposomes encapsulating a bioactive agent, the methodcomprising providing a composition of liposomes prepared with a liposomeloading agent as described above and adding a bioactive agent to thecomposition of liposomes under conditions such that the bioactive agentis transported to the intraliposomal space of the liposomes.

In some embodiments, the present invention provides a liposomecomposition made by the methods described above. In some embodiments,the liposome compositions are used for treatment of disease or conditionin an animal.

Bioactive Agents

In some embodiments, biological substances and/or therapeutic agents(e.g., “drugs”) are incorporated by encapsulation within liposomes(e.g., in the intraliposomal space). Examples of bioactive agentsinclude but are not limited to antianginas, antiarrhythmics,antiasthmatic agents, antibiotics, antimalarials, antidiabetics,antifungals, antihistamines, antihypertensives, antiparasitics,antineoplastics, antivirals, cardiac glycosides, herbicides, hormones,immunomodulators, antibodies (e.g., monoclonal, human, humanized,chimeric, etc., antibodies), neurotransmitters, nucleic acids,pesticides, proteins, radio contrast agents, radionuclides, sedatives,analgesics, steroids, tranquilizers, vaccines, vasopressors,anesthetics, and/or peptides.

The drugs that can be incorporated into the dispersion system astherapeutic agents include chemicals as well as biologics. The term“chemicals” encompasses compounds that are classically referred to asdrugs, such as antitumor agents, anesthetics, analgesics, antimicrobialagents, opiates, hormones, etc.

Of particular interest for inclusion in the liposome compositions of thepresent technology are analgesics, e.g., opiates and/or opioids (e.g.,hydromorphone and buprenorphine), opioid antagonists (e.g., naltrexone),and quinoline drugs (e.g., a 4-aminoquinoline such as chloroquine).

The term “biologics” encompasses nucleic acids (e.g., DNA and RNA),proteins and peptides, and includes compounds such as cytokines,hormones (e.g., pituitary and hypophyseal hormones), growth factors,vaccines, etc.

Suitable antibiotics for inclusion in the liposome compositions of thepresent technology include, but are not limited to, loracarbef,cephalexin, cefadroxil, cefixime, ceftibuten, cefprozil, cefpodoxime,cephradine, cefuroxime, cefaclor, neomycin/polymyxin/bacitracin,dicloxacillin, nitrofurantoin, nitrofurantoin macrocrystal,nitrofurantoin/nitrofuran mac, dirithromycin, gemifloxacin, ampicillin,gatifloxacin, penicillin V potassium, ciprofloxacin, enoxacin,amoxicillin, amoxicillin/clavulanate potassium, clarithromycin,levofloxacin, moxifloxacin, azithromycin, sparfloxacin, cefdinir,ofloxacin, trovafloxacin, lomefloxacin, methenamine, erythromycin,norfloxacin, clindamycin/benzoyl peroxide, quinupristin/dalfopristin,doxycycline, amikacin sulfate, vancomycin, kanamycin, netilmicin,streptomycin, tobramycin sulfate, gentamicin sulfate, tetracyclines,framycetin, minocycline, nalidixic acid, demeclocycline, trimethoprim,miconazole, colistimethate, piperacillin sodium/tazobactam sodium,paromomycin, colistin/neomycin/hydrocortisone, amebicides,sulfisoxazole, pentamidine, sulfadiazine, clindamycin phosphate,metronidazole, oxacillin sodium, nafcillin sodium, vancomycinhydrochloride, clindamycin, cefotaxime sodium, co-trimoxazole,ticarcillin disodium, piperacillin sodium, ticarcillindisodium/clavulanate potassium, neomycin, daptomycin, cefazolin sodium,cefoxitin sodium, ceftizoxime sodium, penicillin G potassium and sodium,ceftriaxone sodium, ceftazidime, imipenem/cilastatin sodium, aztreonam,cinoxacin, erythromycin/sulfisoxazole, cefotetan disodium, ampicillinsodium/sulbactam sodium, cefoperazone sodium, cefamandole nafate,gentamicin, sulfisoxazole/phenazopyridine, tobramycin, lincomycin,neomycin/polymyxin B/gramicidin, clindamycin hydrochloride,lansoprazole/clarithromycin/amoxicillin, alatrofloxacin, linezolid,bismuth subsalicylate/metronidazole/tetracycline, erythromycin/benzoylperoxide, mupirocin, fosfomycin, pentamidine isethionate,imipenem/cilastatin, troleandomycin, gatifloxacin, chloramphenicol,cycloserine, neomycin/polymyxin B/hydrocortisone, ertapenem, meropenem,cephalosporins, fluconazole, cefepime, sulfamethoxazole,sulfamethoxazole/trimethoprim, neomycin/polymyxin B, penicillins,rifampin/isoniazid, erythromycin estolate, erythromycin ethylsuccinate,erythromycin stearate, ampicillin trihydrate, ampicillin/probenecid,sulfasalazine, sulfanilamide, sodium sulfacetamide, dapsone, doxycyclinehyclate, trimenthoprim/sulfa, methenamine mandelate, plasmodicides,pyrimethamine, hydroxychloroquine, chloroquine phosphate, chloroquinediphosphate, trichomonocides, anthelmintics, atovaquone.

Liposome Loading

Liposomes find use in pharmaceutical preparations, e.g., to improve thecharacteristics (e.g., bioavailability, pharmacokinetics, toxicity,etc.) of a drug or other bioactive agent (“pharmaceutical agent”) whenadministered to a patient. In particular, therapies employing bioactiveagents can in many cases be improved by encapsulating the agent inliposomes rather than administering the free agent directly into thebody. For example, incorporation of such agents in liposomes can changetheir activities, clearance rates, tissue distributions, and toxicitiescompared to direct administration. Liposomes themselves have beenreported to have no significant toxicities in previous human clinicaltrials where they have been given intravenously. See, e.g., Richardsonet al., (1979), Br. J. Cancer 40:35; Ryman et al., (1983) in “Targetingof Drugs” G. Gregoriadis, et al., eds. pp 235-248, Plenum, N.Y.;Gregoriadis G., (1981), Lancet 2:241, and Lopez-Berestein et al., (1985)J. Infect. Dis., 151:704. Liposomes are reported to concentratepredominantly in the reticuloendothelial organs lined by sinusoidalcapillaries, e.g., liver, spleen, and bone marrow, and phagocytosed bythe phagocytic cells present in these organs.

When liposomes are used in a liposome drug delivery system, a bioactiveagent such as a drug is entrapped in the liposome and then administeredto the patient to be treated. For example, see Rahman et al., U.S. Pat.No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Paphadjopoulos et al.,U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk etal., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No.4,588,578. Alternatively, if the bioactive agent is lipophilic, it mayassociate with the lipid bilayer. Typically, the term “entrapment”includes both the drug in the aqueous volume of the liposome as well asdrug associated with the lipid bilayer.

Liposome formulations for pharmaceutical applications can be made eitherby combining drug and lipid before formation of the vesicles or by“loading” lipid vesicles with drug after the liposomes have been formed.Upon administration to a patient, liposomes biodistribute and interactwith cells in the body according to route of administration, vesicularcomposition, and vesicular size. Charge, chemistry, and bilayercomponents (e.g., the inclusion on the vesicle surface of protectivepolymers or targeting moieties) all change the way liposomes behave inthe patient.

In some embodiments, the pharmaceutical agent is loaded into pre-formedliposomes using a loading procedure, for example, by using a pHgradient. In some embodiments, the pharmaceutical agent may precipitatein the interior of the liposome. This precipitation protects thepharmaceutical agent and the lipids from degradation (e.g., hydrolysis).In some embodiments, an excipient such as citrate or sulfateprecipitates the pharmaceutical agent and can be utilized in theinterior of the liposomes together with a gradient to promote drugloading.

In some embodiments, liposomal entrapment of bioactive agents iseffected by employing transmembrane ion gradients (see, e.g., Int'l Pat.Appl. PCT/US1985/001501). Aside from inducing uptake, such transmembranegradients also act to increase drug retention in the liposomes. Forexample, transmembrane pH gradients (ΔpH) influence the drug loading ofcertain weak acids and weak bases. See, for example, Jacobs, Quant.Biol. 8:30-39 (1940), Chapper, et al. in Regulation of MetabolicProcesses in Mitochondria, Tager, et al. eds. Elsevier, Amsterdam, pp.293-316 (1966), Crofts, J. Biol. Chem. 242:3352-3359 (1967), Crofts,Regulatory Functions of Biological Membranes, Jarnefelt, ed., ElsevierPublishing Co., Amsterdam, pp. 247-263 (1968), Rottenberg, Bioenergetics7:61-74 (1975), and Rottenberg, Methods in Enzmol. 55:547-569 (1979).This behavior stems from the permeable nature of the neutral forms ofthese molecules, which contrasts with the impermeable nature of thecharged forms. Thus, if a neutral amine (such as ammonia) diffusesacross a biological membrane or vesicle exhibiting a ΔpH (e.g., with anacidic interior), it will become protonated and therefore become trappedin the vesicle interior.

In some particular embodiments, ammonium sulfate is used as a liposomeloading agent (e.g., a liposome loading base) under conditions whereboth the ammonium and the drug are predominantly in the charged (e.g.,protonated) form. For example, the loading of a drug (e.g.,hydromorphone) into ammonium sulfate-loaded liposomes is a function ofthe amount (μmol) of drug (e.g., hydromorphone) added (FIG. 1, solidlines). In particular, the loading efficiency of drug (e.g.,hydromorphone) is approximately 50% for liposomes having anextraliposomal pH of approximately of 3 to 6 and an amount of added drug(e.g., hydromorphone) that is equal to the amount of ammonium in theliposomes (FIG. 1, middle solid line). However, raising the pH of theextraliposomal space to a value (e.g., 11) at which the drug (e.g.,hydromorphone) (having a pKa of approximately 7.8 to 8.9) and theammonia (having a pKa of approximately 9.3) are in the neutral formgreatly diminishes the loading efficiency of drug (e.g., hydromorphone)as a function of the amount of drug (e.g., hydromorphone) added (FIG. 1,bottom solid line). This occurs because the drug (e.g., hydromorphone)is a weaker base than ammonia.

Weak Bases

Based on these observations, this phenomenon is used advantageously toload liposomes by replacing the ammonia with a substance that is aweaker base than the drug, e.g., a weak base such as pyridine ornicotinamide. Similar to the loading of ammonium sulfate-loadedliposomes, loading of a drug (e.g., hydromorphone) into liposomes loadedwith a weak base salt, e.g., the sulfate salt of pyridine (having a pKaof approximately 5.3), is a function of the amount of drug (e.g.,hydromorphone) added. And, when the pH of the extraliposomal compartmentis 3, the loading is the same or substantially the same as the loadingobserved with ammonium sulfate-loaded liposomes because both the drug(e.g., hydromorphone) and the weak base (e.g., pyridine or nicotinamide)are predominantly in the charged form, even in the extraliposomalcompartment (FIG. 1, middle solid line). However, as the pH of theextraliposomal compartment is raised (e.g., to 11), drug (e.g.,hydromorphone) loading is elevated to near quantitative levels (e.g.,nearly all drug (e.g., hydromorphone) is loaded) provided that theamount of drug (e.g., hydromorphone) added is less than the amount ofweak base (e.g., pyridinium) ions inside the liposomes (see, e.g., FIG.1, upper dashed line).

In addition to improving drug loading, using a weaker base for loadingalso reduces the initial rapid release rate of the drug that is observedwhen ammonium sulfate is used for loading. Early phase leakage is atransient, self-limiting process because leakage of a base in theneutral form lowers the internal pH of the liposomes, which in turnreduces the concentration of neutral bases. Ammonia has a log Pestimated to be approximately −0.3 and a pKa of 9.3. Therefore, in thisexemplary composition for loading a drug (e.g., hydromorphone) whose logP is 1.3 and pKa is approximately 7.8 to 8.9, the drug is released fromthe liposomes more than the residual ammonia. In contrast, a loadingbase that is a weaker base than the drug to be loaded (e.g., inparticular, a weak base having a (log P−pKa) that is higher than the(log P−pKa) of the drug) leaks more from the liposomes than the drug.Accordingly, using a base for drug loading that is a weaker base thanthe drug and/or has a (log P−pKa) greater than the (log P−pKa) of thedrug eliminates or minimizes the early rapid release of drug from theliposomes.

The technology is not limited in the weak base used for loadingliposomes. Exemplary weak bases include pyridine, 2-methoxypyridine,pyridazine, adenine, aniline, and nicotinamide and derivatives thereof.Furthermore, the technology is not limited in the phase of the weak baseused for loading liposomes. For example, both adenine (solid at roomtemperature) and aniline (liquid at room temperature) find use inembodiments of the technology.

Pyridine is a heterocyclic organic base having a pKa of 5.3. Pyridine isa liquid that is miscible with water at all ratios. Pyridine ishydrophobic in the neutral form, with an estimated log P ofapproximately 1.3. When added to a strong acid solution, pyridine isprotonated to form a pyridinium salt; the pH varies over the range of 3to 7 depending on the proportion of pyridine to acid used.

2-methoxypyridine is a pyridine derivative having a pKa of 3.28.2-methoxypyridine is a liquid with limited solubility in water.2-methoxypyridine is hydrophobic in the neutral form, with an estimatedlog P of approximately 1.3 (similar to pyridine). When added to a strongacid solution, 2-methoxypyridine is protonated to form a highly soluble2-methoxypyridinium salt; the pH varies over the range of 1 to 4depending on the proportion of 2-methoxypyridine to acid used.

Pyridazine is a heterocyclic organic base having a pKa of 2.33.Pyridazine is a liquid that is miscible with water at all ratios. Incontrast to pyridine, pyridazine is hydrophilic in the neutral form,with an estimated log P of approximately −0.7. When added to a strongacid solution, pyridazine is protonated to form a pyridazinium salt; thepH varies over the range of 1 to 3 depending on the proportion ofpyridazine to acid used.

Adenine (6-aminopurine) is a purine derivative having a pKa of 4.15.Adenine is a solid that forms soluble salts with strong acids, e.g.,perchloric and triflic acids.

Aniline (phenylamine, aminobenzene) is an organic compound with a pKa of4.19. Aniline is a hydrophobic liquid that reacts with strong acids toform anilinium (phenylammonium) ions and that forms soluble salts withstrong acids, e.g., triflic acid.

In some embodiments, a weak base and a strong acid are used to produce aweak base salt. As used herein, the term “strong acid” refers to an acidthat ionizes (e.g., in an aqueous solution) completely by losing oneproton, e.g., in some embodiments, a strong acid is an acid that isstronger in aqueous solution than a hydronium ion—accordingly, in someembodiments, strong acids are acids with a pKa less than approximately−1.74. In addition, the term “strong acid” as used herein refers toacids that dissociate nearly completely in very dilute solution thoughthey may or may not be more acidic than hydronium ion. The term “strongacid” as used herein refers also to extremely strong acids (e.g.,fluoroantimonic acid, triflic acid) and superacids that protonate waterto give ionic, crystalline hydronium “salts”, e.g., fluoroantimonicacid, magic acid, and perchloric acid. Accordingly, non-limitingexamples of strong acids are, e.g., sulfuric acid (first dissociationonly), hydrochloric acid, nitric acid, hydrobromic acid, hydroiodicacid, triflic acid, perchloric acid, bromic acid, perbromic acid, iodicacid, periodic acid, and sulfonic acids (organic oxyacids) such as,e.g., methanedisulfonic acid, methanesulfonic acid, p-toluenesulfonicacid (tosylic acid), 1,2-ethanedisulfonic acid (“edisylate”), and1,3-propanedisulfonic acid (“eprodisate”).

In particular embodiments, the strong acid is a diprotic acid havingboth first and second pKa values less than zero. Furthermore, it iscontemplated that minimizing the molecular weight of the acid andmaximizing the polarity of the acid provides additional guidance forselecting a suitable acid for embodiments of the technology.

Embodiments relate to the use of a weak base. As used herein, a weakbase is a chemical base that does not completely ionize (e.g., in anaqueous solution), e.g., a chemical base that is partially protonated(e.g., in an aqueous solution).

Embodiments relate to the use of a weak base salt, e.g., a salt producedby the ionization of a weak base, e.g., by a strong acid.

In particular embodiments, a composition comprising liposomes is madeusing a weak base salt solution having a pH of at least 2, e.g., toallow for swelling of phospholipid (e.g.,dipalmitoylphosphatidylcholine) during incubation (e.g., at 35 to 55°C., e.g., approximately 40° C., e.g., 42° C.). It is further preferredthat the pH of the weak base salt solution is below the pKa of the weakbase to limit the amount of free base in the solution, thus stabilizingthe liposome membranes. In some embodiments, liposomes are prepared inthe base salt solution and excess weak base is eliminated by standardwashing techniques (e.g., sedimentation in a centrifuge, dialysis, gelchromatography, etc.). Then, a drug, bioactive agent, pharmaceuticalagent, etc. is added to the composition comprising liposomes. Incubatingthe composition comprising the drug and the liposomes for a period of atleast 30 minutes to 72 hours (e.g., at least 30 minutes, 1 hour, 1.5hours, 2 hours, 2.5 hours, 3.0 hours, 3.5 hours, 4 hours, 4.5 hours, 5hours, 12 hours, 24 hours, 48 hours, 72 hours or from 6 hours to 24hours, 12 hours to 24 hours, 24 to 48 hours, 6 hours to 72 hours, 12hours to 72 hours or 24 to 72 hours) produces a composition comprisingliposomes loaded with the drug (e.g., the drug moves into the liposomes(e.g., into the intraliposomal space)). Manipulation and control of theextraliposomal (e.g., bulk) phase pH provides for control of drugloading (e.g., to maximize loading (e.g., to maximize efficiency of drugloading)). For example, in some embodiments a buffer is provided in theextraliposomal phase to control the extraliposomal pH. For example, insome embodiments a buffer is added to particular compositions tomaintain the extraliposomal pH at a value at which the drug to be loadedis predominantly in the protonated (e.g., charged) form. For example,for drugs that are weak bases, the protonated form is a charged form.

In some embodiments, the preferable external loading pH for pyridinesalts is 6 to 8; further, in some embodiments, the external loading pHis lower than 6 for compositions comprising 2-methoxypyridine orpyridazine (but greater than the pKa of the 2-methoxypyridine orpyridazine).

During the development of the technology described herein, experimentswere performed indicating that the drugs chloroquine, doxycycline,hydromorphone, naltrexone, and buprenorphine are loaded into liposomeswith high efficiency using one or more weak base compounds (e.g.,pyridine, 2-methoxypyridine, pyridazine) In particular, loadingliposomes using a weak base provided an improved (e.g., increased, e.g.,high) loading efficiency and an improved (e.g., reduced, e.g., very low)in vitro leakage of the drug from the liposomes in the early phase ofleakage (0 to 48 hours). The data collected indicated that liposomeloading with a weak base is much improved to liposomes loaded usingammonium sulfate loading. In some embodiments, leakage of the loadingbase itself is quite rapid during the first few days. Thus, in someembodiments the weak loading bases act as sacrificial leakage agents,thereby eliminating the early phase loss of drug from liposomes. Thatis, in the early rapid phase of leakage from loaded liposomes, the weakbase leaks from the liposomes in lieu of the drug leaking from theliposomes, thus maintaining a high amount of drug inside the liposomes.

Pharmaceutical Preparations

In some embodiments, liposome compositions prepared by the methodsdescribed herein are administered alone or in a mixture with aphysiologically-acceptable carrier (such as physiological saline orphosphate buffer) selected in accordance with the route ofadministration and standard pharmaceutical practice. Generally, normalsaline is employed as the pharmaceutically acceptable carrier. Othersuitable carriers include, e.g., water, buffered water, 0.4% saline,0.3% glycine, and the like, including glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc. In compositionscomprising saline or other salt-containing carriers, the carrier ispreferably added following liposome formation. Thus, after the liposomeis formed and loaded with a suitable drug, the liposome can be dilutedinto pharmaceutically acceptable carriers such as normal saline. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with a sterile aqueous solution prior toadministration. The compositions may also contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH-adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.

Additionally, the composition may include lipid-protective agents thatprotect lipids against free-radical and lipid-peroxidative damages onstorage. Lipophilic free-radical quenchers, such as alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, aresuitable.

The concentration of liposomes in the pharmaceutical formulations canvary widely, e.g., from less than approximately 0.05%, usually at leastapproximately 2 to 5% to as much as 10 to 30% by weight and are selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, liposomes composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration. The amount of liposomes administered will depend uponthe particular drug used, the disease state being treated and thejudgment of the clinician but will generally be between approximately0.01 and approximately 50 mg per kilogram of body weight, preferablybetween approximately 0.1 and approximately 5 mg per kg of body weight.

In some embodiments, it is desirable to include polyethylene glycol(PEG)-modified phospholipids, PEG-ceramide, or gangliosideG_(M1)-modified lipids to the liposomes. Addition of such componentsprevents liposome aggregation and provides for increasing circulationlifetime and increasing the delivery of the loaded liposomes to thetarget tissues. Typically, the concentration of the PEG-modifiedphospholipids, PEG-ceramide, or G_(M1)-modified lipids in the liposomewill be approximately 1 to 15%.

In some embodiments, overall liposome charge is an important determinantin liposome clearance from the blood. Charged liposomes are typicallytaken up more rapidly by the reticuloendothelial system (Juliano,Biochem. Biophys. Res. Commun. 63: 651 (1975)) and thus have shorterhalf-lives in the bloodstream. Liposomes with prolonged circulationhalf-lives are typically desirable for therapeutic and certaindiagnostic uses. For instance, liposomes that are maintained from 8, 12,or up to 24 hours in the bloodstream are particularly preferred.

In another example of their use, drug-loaded liposomes can beincorporated into a broad range of topical dosage forms including butnot limited to gels, oils, emulsions, and the like. For instance, insome embodiments the suspension containing the drug-loaded liposomes isformulated and administered as a topical cream, paste, ointment, gel,lotion, and the like.

The present technology also provides liposome compositions in kit form.The kit will typically comprise a container that is compartmentalizedfor holding the various elements of the kit. The kit contains thecompositions of the present inventions, preferably in dehydrated form,with instructions for their rehydration and administration.

In still other embodiments, the drug-loaded liposomes have a targetingmoiety attached to the surface of the liposome. Methods of attachingtargeting moieties (e.g., antibodies, proteins) to lipids (such as thoseused in the present particles) are known to those of skill in the art.

Dosage for the drug-loaded liposome formulations depends on the ratio ofdrug to lipid and the administrating physician's and/or veterinarian'sopinion based on age, weight, and condition of the patient.

In some embodiments, compositions comprising liposomes encapsulating abioactive agent are formulated with a buffering agent. The bufferingagent may be any pharmaceutically acceptable buffering agent. Buffersystems include citrate buffers, acetate buffers, borate buffers, andphosphate buffers. Examples of buffers include citric acid, sodiumcitrate, sodium acetate, acetic acid, sodium phosphate and phosphoricacid, sodium ascorbate, tartaric acid, maleic acid, glycine, sodiumlactate, lactic acid, ascorbic acid, imidazole, sodium bicarbonate andcarbonic acid, sodium succinate and succinic acid, histidine, and sodiumbenzoate and benzoic acid.

In some embodiments, compositions comprising liposomes encapsulating abioactive agent are formulated with a chelating agent. The chelatingagent may be any pharmaceutically acceptable chelating agent. Chelatingagents include ethylenediaminetetraacetic acid (also synonymous withEDTA, edetic acid, versene acid, and sequestrene), and EDTA derivatives,such as dipotassium edetate, disodium edetate, edetate calcium disodium,sodium edetate, trisodium edetate, and potassium edetate. Otherchelating agents include citric acid and derivatives thereof. Citricacid also is known as citric acid monohydrate. Derivatives of citricacid include anhydrous citric acid and trisodiumcitrate-dihydrate. Stillother chelating agents include niacinamide and derivatives thereof andsodium deoxycholate and derivatives thereof.

In some embodiments, compositions comprising liposomes encapsulating abioactive agent are formulated with an antioxidant. The antioxidant maybe any pharmaceutically acceptable antioxidant. Antioxidants are wellknown to those of ordinary skill in the art and include materials suchas ascorbic acid, ascorbic acid derivatives (e.g., ascorbylpalmitate,ascorbylstearate, sodium ascorbate, calcium ascorbate, etc.), butylatedhydroxy anisole, buylated hydroxy toluene, alkylgallate, sodiummeta-bisulfate, sodium bisulfate, sodium dithionite, sodiumthioglycollic acid, sodium formaldehyde sulfoxylate, tocopherol andderivatives thereof, (d-alpha tocopherol, d-alpha tocopherol acetate,dl-alpha tocopherol acetate, d-alpha tocopherol succinate, betatocopherol, delta tocopherol, gamma tocopherol, and d-alpha tocopherolpolyoxyethylene glycol 1000 succinate) monothioglycerol, and sodiumsulfite. Such materials are typically added in ranges from 0.01 to 2.0%.

In some embodiments, compositions comprising liposomes encapsulating abioactive agent are formulated with a cryoprotectant. The cryoprotectingagent may be any pharmaceutically acceptable cryoprotecting agent.Common cryoprotecting agents include histidine, polyethylene glycol,polyvinyl pyrrolidine, lactose, sucrose, mannitol, and polyols.

In some embodiments, compositions comprising liposomes encapsulating abioactive agent are formulated with an isotonicity agent. Theisotonicity agent can be any pharmaceutically acceptable isotonicityagent. This term is used in the art interchangeably with iso-osmoticagent, and is known as a compound that is added to the pharmaceuticalpreparation to increase the osmotic pressure, e.g., in some embodimentsto that of 0.9% sodium chloride solution, which is iso-osmotic withhuman extracellular fluids, such as plasma. Preferred isotonicity agentsare sodium chloride, mannitol, sorbitol, lactose, dextrose and glycerol.

Compositions of the liposomes encapsulating a bioactive agent mayoptionally comprise a preservative. Common preservatives include thoseselected from the group consisting of chlorobutanol, parabens,thimerosol, benzyl alcohol, and phenol. Suitable preservatives includebut are not limited to: chlorobutanol (0.3-0.9% w/v), parabens(0.01-5.0%), thimerosal (0.004-0.2%), benzyl alcohol (0.5-5%), phenol(0.1-1.0%), and the like.

In some embodiments, compositions comprising liposomes encapsulating abioactive agent are formulated with a humectant to provide a pleasantmouth-feel in oral applications. Humectants known in the art includecholesterol, fatty acids, glycerin, lauric acid, magnesium stearate,pentaerythritol, and propylene glycol.

In some embodiments, an emulsifying agent is included in theformulations, for example, to ensure complete dissolution of allexcipients, especially hydrophobic components such as benzyl alcohol.Many emulsifiers are known in the art, e.g., polysorbate 60.

For some embodiments related to oral administration, it may be desirableto add a pharmaceutically acceptable flavoring agent and/or sweetener.Compounds such as saccharin, glycerin, simple syrup, and sorbitol areuseful as sweeteners.

Administration and Therapy

Once the therapeutic agent has been loaded into the liposomes, thecombination can be administered to a patient by a variety of techniques.

Preferably, the pharmaceutical compositions are administeredparenterally, e.g., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection. For example, see Raham et al.,U.S. Pat. No. 3,993,754; Sears, U.S. Pat. No. 4,145,410; Papahadjopouloset al., U.S. Pat. No. 4,235,871; Schneider, U.S. Pat. No. 4,224,179;Lenk et al., U.S. Pat. No. 4,522,803; and Fountain et al., U.S. Pat. No.4,588,578. Particular formulations that are suitable for this use arefound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed. (1985). Typically, the formulations comprisea solution of the liposomes suspended in an acceptable carrier,preferably an aqueous carrier. A variety of aqueous carriers are used inembodiments of the technology, e.g., water, buffered water, 0.9%isotonic saline, and the like. These compositions may be sterilized byconventional, well-known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

Dosage for the liposome formulations will depend on the ratio of drug tolipid and the administrating physician's opinion based on age, weight,and condition of the patient.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open”, or “closed”procedures. By “topical”, it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures include incising the skin of a patientand directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrizamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The compositions of the present invention that further comprise atargeting antibody on the surface of the liposome are particularlyuseful for the treatment of certain diseases.

The therapeutic use of liposomes can include the delivery of drugs thatare normally toxic in the free form. In the liposomal form, the toxicdrug may be directed away from the sensitive tissue where toxicity canresult and targeted to selected areas where they can exert theirtherapeutic effects. Liposomes can also be used therapeutically torelease drugs slowly, over a prolonged period of time, thereby reducingthe frequency of drug administration through an enhanced pharmacokineticprofile. In addition, liposomes can provide a method for forming anaqueous dispersion of hydrophobic drugs for intravenous delivery.

The route of delivery of liposomes can also affect their distribution inthe body. Passive delivery of liposomes involves the use of variousroutes of administration e.g., parenterally, although other effectiveadministration forms, such as intraarticular injection, inhalant mists,orally active formulations, transdermal iotophoresis, or suppositoriesare also envisioned. Each route produces differences in localization ofthe liposomes.

Because dosage regimens for pharmaceutical agents are well known tomedical practitioners, the amount of the liposomal pharmaceutical agentformulations that is effective or therapeutic for the treatment of adisease or condition in mammals and particularly in humans will beapparent to those skilled in the art. The optimal quantity and spacingof individual dosages of the formulations herein will be determined bythe nature and extent of the condition being treated, the form, routeand site of administration, and the particular patient being treated,and such optima can be determined by conventional techniques. It willalso be appreciated by one of skill in the art that the optimal courseof treatment, e.g., the number of doses given per day for a definednumber of days, can be ascertained by those skilled in the art usingconventional course of treatment determination tests.

The liposomes containing therapeutic agents and the pharmaceuticalformulations thereof of the present technology and those produced by theprocesses thereof can be used therapeutically in animals (including man)in the treatment of infections or conditions which require: (1) repeatedadministrations, (2) the sustained delivery of the drug in its bioactiveform, or (3) the decreased toxicity with suitable efficacy compared withthe free drug in question.

The mode of administration of the liposomes containing thepharmaceutical agents and the pharmaceutical formulations thereofdetermine the sites and cells in the organism to which the compound willbe delivered. The liposomes of the present technology can beadministered alone but will generally be administered in admixture witha pharmaceutical carrier selected with regard to the intended route ofadministration and standard pharmaceutical practice. The preparationsmay be injected parenterally, for example, intravenously. For parenteraladministration, they can be used, for example, in the form of a sterileaqueous solution that may contain other solutes, for example, enoughsalts or glucose to make the solution isotonic.

For the oral mode of administration, the liposomal therapeutic drugformulations of this technology can be used in the form of tablets,capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutionsand suspensions, and the like. In the case of tablets, carriers that canbe used include lactose, sodium citrate, and salts of phosphoric acid.Various disintegrants, such as starch, and lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc, are commonly used intablets. For oral administration in capsule form, useful diluents arelactose and high molecular weight polyethylene glycols. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweeteningand/or flavoring agents can be added.

For the topical mode of administration, the liposome-drug formulationsof the present technology may be incorporated into dosage forms such asgels, oils, emulsions, and the like. Such preparations may beadministered by direct application as a cream, paste, ointment, gel,lotion or the like.

For administration to humans in the curative, remissive, retardive, orprophylactic treatment of diseases the prescribing physician willultimately determine the appropriate dosage of the drug for a givenhuman subject, and this can be expected to vary according to the age,weight, and response of the individual as well as the nature andseverity of the patient's disease. The dosage of the drug in liposomalform will generally be approximately that employed for the free drug. Insome cases, however, it may be necessary to administer dosages outsidethese limits and, in some embodiments, the technology comprisesadministering dosages in excess of these limits due to theextended-release characteristics of the formulations.

The term “therapeutically effective” as it pertains to the compositionsof the invention means that a biologically active substance present inthe aqueous component within the vesicles is released in a mannersufficient to achieve a particular medical effect for which thetherapeutic agent is intended. Examples, without limitation, ofdesirable medical effects that can be attained are chemotherapy,antibiotic therapy, and regulation of metabolism. Exact dosages willvary depending upon such factors as the particular therapeutic agent anddesirable medical effect, as well as patient factors such as age, sex,general condition, and the like. Those of skill in the art can readilytake these factors into account and use them to establish effectivetherapeutic concentrations without resort to undue experimentation.

Generally, however, the dosage range appropriate for human use includesthe range of 0.1 to 6000 mg/m² of body surface area. For someapplications, such as intravenous administration, the dose required maybe quite small, but for other applications, such as subcutaneous and/orintraperitoneal administration, the dose desired to be used may be verylarge. While doses outside the foregoing dose range may be given, thisrange encompasses the breadth of use for practically all thebiologically active substances.

The liposomes may be administered for therapeutic applications by anydesired route, for example, intramuscular, intraarticular, epidural,intrathecal, intraperitoneal, subcutaneous, intravenous, intralymphatic,oral and submucosal, and by implantation under many different kinds ofepithelia, including the bronchialar epithelia, the gastrointestinalepithelia, the urogenital epithelia, and various mucous membranes of thebody.

In addition, the liposomes of the invention can be used to encapsulatecompounds useful in agricultural applications, such as fertilizers,pesticides, and the like. For use in agriculture, the liposomes can besprayed or spread onto an area of soil where plants will grow and theagriculturally effective compound contained in the vesicles will bereleased at a controlled rate by contact with rain and irrigationwaters. Alternatively the slow-releasing vesicles can be mixed intoirrigation waters to be applied to plants and crops. One skilled in theart will be able to select an effective amount of the compound useful inagricultural applications to accomplish the particular goal desired,such as the killing of pests, the nurture of plants, etc.

During the development of embodiments of the technology provided,experiments were conducted to collect data relevant to the in vivo useof the liposome preparations and delivery of pharmaceuticals to asubject. See, e.g., Example 9. In particular, during the experiments apharmaceutical (e.g., buprenorphine) was loaded into liposomes accordingto embodiments of the technology provided herein, e.g., using a weakbase (e.g., 2-methoxypyridinium sulfate and/or 2-methoxypyridiniumeprodisate). Data collected indicated that weak base loading providestherapeutically significant serum concentrations of the pharmaceuticalfor a longer in vivo period of time than the period of time thattherapeutically significant serum concentrations of the pharmaceuticalare provided by present techniques such as acid loading. Furthermore,the data indicated that the technology (e.g., the use of eprodisate)provides serum concentrations of the pharmaceutical during the first twoweeks after administration that are lower than the serum concentrationsof the pharmaceutical when administered using present technologies. Inparticular, present technologies generally provide serum concentrationsof pharmaceuticals that are higher than needed to provide an effect,thus resulting in inefficient use of the pharmaceutical and/or providingthe pharmaceutical a level that produces undesired side effectsassociated with unnecessarily elevated dosages. Accordingly, thetechnology described herein provides an improved effective dose of apharmaceutical (e.g., the dose is not too high and/or the dose is nothigher than is needed to provide an effect) relative to presenttechnologies.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation.

EXAMPLES

During the development of embodiments of the technologies describedherein, experiments were conducted to evaluate the loading of drugs intoliposomes using weak loading bases as described herein. In particular,data were collected to measure the efficiency of drug loading intoliposomes loaded with a weak loading base and data were collected tomeasure drug and loading base leakage from the loaded liposomes into theextraliposomal space.

Methods

Spectrophotometric Analysis. Concentrations of loading bases and drugsloaded were measured using characteristic UV spectra and knownabsorbances at defined wavelengths. To determine the concentrations ofthe drug and the loading base in compositions comprising both the drugand the loading base, readings at two wavelengths were used and theconcentrations of the drug and the loading base were solved usingsimultaneous equations. Reference values for the extinction coefficientsof each substance at both reference wavelengths were determined.

Liposome Preparation. A mixture containing 80 μmoldipalmitoylphosphatidylcholine (DPPC) and 40 μmol cholesterol (chol) wasdried from a chloroform solution using a rotary evaporator. The lipidmixture was resuspended in 1 mL of tert-butanol, transferred to a 16-mmscrew cap tube and frozen in a slurry of dry ice and 2-propanol. Thetube was placed in a lyophilizer flask and freeze dried for 24 hours toproduce a microporous lipid lyophilizate. Lipid lyophilizates werestored at −20° C. until use. Prior to liposome preparation, the lipidlyophilizate was removed from the freezer and allowed to warm to 22° C.A 1-mL aliquot of loading base solution was added to the lipidlyophilizate and the mixture was swollen in a water bath at 55° C. for10 minutes to form liposomes. To eliminate the excess loading base, theliposome suspension was cooled to 22° C., diluted with isotonic NaCl,and sedimented at 300×g for 10 minutes. After aspiration of thesupernatant, the pellet was resuspended two times in isotonic NaClfollowed by sedimentation at 300×g for 10 minutes. The final liposomepellet was resuspended in 1 mL of an appropriate loading medium. Variousbuffer choices were used for the loading medium as indicated below forthe specific drug to be loaded in each experiment. Then, a sample wassolubilized with 1:3:1 v/v/v chloroform:methanol:water and analyzedspectrophotometrically to measure loading base content. Drug for loadingwas added to the suspension and incubated at 22° C. for 20 hours.Unencapsulated drug and released loading base were removed by dilutingthe solution 3 times with 8 mL of isotonic NaCl followed bysedimentation at 300×g for 10 minutes. After the third wash, theliposome pellet was suspended in isotonic NaCl solution. An aliquot ofthe suspension was removed from each preparation and solubilized in1:3:1 v/v/v chloroform:methanol:water, and the amounts of drug andloading base in the liposomes were quantified spectrophotometrically.

Liposome Leakage, Dialysis Method. A 0.5-mL aliquot of liposomesuspension was placed in a section of dialysis tubing tied at one end.The tubing was tied at the other end to form a sealed bag. The bag wasplaced in 4.5 mL of isotonic NaCl saline solution and placed on anorbital shaker. At specific time points, the dialysis bags wereexchanged into fresh isotonic NaCl and the drug and loading base werequantified spectophotometrically in the external dialysis solutions.

Liposome Leakage, Sedimentation Method. Liposome samples were diluted to3-5 mL and left on an orbital shaker at 22° C. At specific time points,the liposomes were sedimented in a centrifuge at 300×g for 5 minutes andan aliquot of the clear supernatant was analyzed spectrophotometricallyfor drug and loading base content. After measurement, the solution wasreturned to the liposome suspension, which was vortexed and returned tothe orbital shaker.

Example 1: Chloroquine

During the development of embodiments of the technology provided herein,experiments were conducted to assess the loading of a chloroquine intoliposomes using weak loading bases, e.g., pyridinium sulfate andpyridinium chloride.

Example 1a: Chloroquine Loaded Using 0.5 M Pyridinium Sulfate

Loading base solution: 0.5 M pyridinium sulfate (1 M with respect topyridine). Loading medium: 220 mM tris-nitrate buffer, pH 8.3.

Base content prior to drug loading: 26.7 μmol base per 80 μmol DPPC. 6.7μmol per loading.

Loading efficiency: Loading of 2.5 μmol and 5 μmol chloroquine was closeto 100% (Table 1). Based on the amount loaded when either 10 or 20 μmolof chloroquine was added, the loading capacity was approximately 9.5μmol.

TABLE 1 Loading of chloroquine into liposomes with 0.5M pyridiniumsulfate chloroquine chloroquine added captured (μmol) (%) 20 55.95 1096.32 5 99.84 2.5 99.83

Leakage: Leakage of chloroquine (FIG. 2) and pyridine (FIG. 3) fromliposomes was followed using the dialysis method. The 2.5-μmol and5-μmol chloroquine preparations show leakage that is linear without aninitial rapid phase of leakage (FIG. 2). The other preparations ofliposomal chloroquine show some elevated leakage in the first 24 hours,though the amount is low (e.g., approximately 0.5 to 2%) (FIG. 2).Long-term chloroquine leakage rates are approximately 0.36% per day(FIG. 2). Based on previous results for ammonium sulfate-loadedliposomes, these small amounts of leakage are consistent with in vivorelease times of at least 3 to 4 weeks in mammals. In contrast, pyridineleakage from the liposomes was initially very rapid for the liposomesloaded with 2.5 or 5 μmol of chloroquine and slower after 3 days (FIG.3). This suggests that during the early phase of leakage, pyridinerather than chloroquine is lost from these liposomes. Thus, pyridineacts as a sacrificial leakage agent. Pyridine loss from liposomes loadedwith 10 or 20 μmol of chloroquine was minimal (FIG. 3), indicating thatmost of the pyridine is displaced from these liposomes in the process ofchloroquine loading.

Example 1b: Chloroquine Loaded Using 1 M Pyridinium Chloride

Loading base solution: 1 M pyridinium chloride (1 M with respect topyridine).

Loading medium: 220 mM tris-nitrate buffer, pH 8.3.

Base content prior to drug loading: 30.2 mol per 80 μmol DPPC. 7.5 μmolper loading.

Loading efficiency: Loading efficiency of 2.5 or 5 μmol chloroquine wasclose to 100% (Table 2). Based on the amount loaded when either 10 or 20μmol of chloroquine was added, the loading capacity was approximately 8μmol.

TABLE 2 Loading of chloroquine into liposomes with 1M pyridiniumchloride chloroquine chloroquine added captured (μmol) (%) 20 44.65 1079.64 5 99.04 2.5 99.35

Leakage. Leakage of chloroquine (FIG. 4) and pyridine (FIG. 5) fromliposomes was followed using the dialysis method. The 2.5-μmol and5-μmol preparations show chloroquine leakage that is linear without aninitial rapid phase of leakage (FIG. 4). The other preparations ofliposomal chloroquine show some elevated leakage in the first 24 hours,though the amount is low (approximately 2 to 4%) (FIG. 4). Long-termchloroquine leakage rates are approximately 0.55% per day (FIG. 4).Based on previous results for ammonium sulfate-loaded liposomes, thesesmall amounts of leakage are consistent with in vivo release times of atleast 3 to 4 weeks in mammals. In contrast, pyridine leakage from theliposomes was initially very rapid for the liposomes loaded with 2.5 or5 μmol of chloroquine and slower after 3 days (FIG. 5). This suggeststhat during the early phase of leakage, pyridine rather than chloroquineis lost from these liposomes. Thus, pyridine acts as a sacrificialleakage agent. Pyridine loss from liposomes loaded with 10 or 20 μmol ofchloroquine was minimal (FIG. 5), demonstrating that most of thepyridine is displaced from these liposomes in the process of chloroquineloading.

Comparison Example 1c: Chloroquine Loaded Using 0.133 M Ammonium Sulfate

During the development of embodiments of the technology provided herein,experiments were conducted to compare loading efficiency and leakage ofliposomes loaded with drug according to the technology provided hereinwith liposomes loaded with drug according to existing (e.g., ammoniumsulfate) methods.

Leakage. As an indication of the improvements provided by the technologydescribed herein, the results shown in Examples 1a and 1b were comparedto the leakage of chloroquine from liposomes loaded using an existingammonium sulfate gradient method. Leakage was followed using thedialysis method. The data indicated that the initial leakage from theliposomes was extensive and leakage accounted for 18% of the loaded drugin the first 24 hours (FIG. 6). The long-term leakage (e.g., after 72hours) from these liposomes is comparable to that seen with thepyridinium sulfate-loaded liposomes shown above. Accordingly, the majordifferences for pyridinium sulfate loading are the quantitative loadingefficiency and the elimination and/or minimization of rapid short-termleakage.

Example 2: Hydromorphone Loaded Using 0.5 M Pyridinium Sulfate

During the development of embodiments of the technology provided herein,experiments were conducted to assess the loading of a hydromorphone intoliposomes using weak loading bases, e.g., pyridinium sulfate.

Loading base solution: 0.5 M pyridinium sulfate (1 M with respect topyridine).

Loading medium: 220 mM tris-chloride buffer, pH 8.3.

Base content prior to drug loading: 121 μmol in 160 μmol DPPC. 24.58μmol per loading

Loading Efficiency: Loading efficiency of hydromorphone was 43 to 83%(Table 3), depending on the amount added. Based on the amount loadedwhen 48 μmol of hydromorphone was added, the loading capacity wasapproximately 20 μmol.

TABLE 3 Loading of hydromorphone into liposomes with 0.5M pyridiniumsulfate hydromorphone hydromorphone added captured (μmol) (%) 48 42.2824 72.29 12 82.64 6 75.55

Leakage. Leakage of hydromorphone (FIG. 7) and pyridine (FIG. 8) wasfollowed using the sedimentation method. The leakage of hydromorphonefrom these liposomes following loading was very low, e.g., amounting toonly 4 to 11% of the liposome contents after 550 hours (FIG. 7). Thepreparation containing the least drug (6 μmop appears to give an initialburst of leakage, while all others release drug at a steady rate that isas low as 0.1% per day (FIG. 7).

Example 3: Loading of Naltrexone into Liposomes with 1.5 M PyridiniumSulfate

During the development of embodiments of the technology provided herein,experiments were conducted to assess the loading of a naltrexone intoliposomes using weak loading bases, e.g., pyridinium sulfate.

Loading base solution: 1.5 M pyridinium sulfate (3 M with respect topyridine).

Loading medium: 220 mM tris-chloride buffer, pH 7.0, adjusted to pH 8.1

Base content prior to drug loading: 144 μmol per 80 μmol DPPC. 144 μmolper loading.

Loading efficiency: Loading efficiency for 50 μmol naltrexone was 91.7%.This is better than seen with hydromorphone and may reflect the higheramount of pyridine in the liposomes from using 1.5 M pyridinium sulfate.

Leakage. The loss of naltrexone from these liposomes following loadingwas followed using the sedimentation method (FIG. 8). The release ofnaltrexone was uniform as a function of elapsed time and occurredwithout an early rapid release rate, although there is an initialrelease of approximately 4% at 24 hours (FIG. 8). 20% of the liposomecontents are released in 672 hours, a release rate of less than 1% perday (FIG. 8). This rate is faster than was seen for hydromorphone andmay reflect the lower pKa of naltrexone (pKa 7.3) with respect tohydromorphone (pKa 7.8 to 8.9).

Example 4: Buprenorphine

During the development of embodiments of the technology provided herein,experiments were conducted to assess the loading of a buprenorphine intoliposomes using weak loading bases, e.g., pyridinium sulfate,2-methoxypyridinium sulfate, 2-methoxypyridinium triflate, andpyridazinium triflate.

Example 4a: Buprenorphine Loaded Using 1.5 M Pyridinium Sulfate

Loading base solution: 1.5 M pyridinium sulfate (3 M with respect topyridine).

Loading medium: 200 mM sodium acetate buffer, pH 3.5 to 4.0.

Base content prior to drug loading: 92 μmol per 80 μmol DPPC. 92 μmolper loading.

Loading efficiency: Loading efficiency for 17.86 μmol buprenorphine was44.47% (Table 5, Example 4a). Buprenorphine has quite limited solubilityowing to its high hydrophobicity. Accordingly, the loading process wasinitiated by loading at pH 3.5 in acetate buffer and slowly increasingthe pH to 4.0 as the loading progressed. These data indicate that, insome embodiments, efficient loading is provided by matching thephysicochemical properties of the loading base (e.g., pyridine) and drug(e.g., buprenorphine). For example, while the data indicate that, ingeneral, efficient loading using pyridine occurs at an extraliposomal pHof at least 7, buprenorphine has very limited solubility at thisparticular pH.

Leakage. The loss of buprenorphine from these liposomes followingloading was followed using the sedimentation method (FIG. 9, Example4a). 22% of the liposome contents were released after approximately 500hours, although the initial release rate was faster with 17% release inthe first 170 hours. This faster release rate may occur becausebuprenorphine is a very hydrophobic drug, e.g., buprenorphine has a logP of 4.3 compared with 1.3 for hydromorphone and naltrexone. This higherlog P may indicate that an initial faster release of buprenorphine isnot prevented by the sacrificial release of pyridine.

Example 4b: Buprenorphine Loaded Using 0.5 M 2-Methoxypyridinium Sulfate

Loading base solution: 0.5 M 2-methoxypyridinium sulfate (1 M withrespect to 2-methoxypyridine).

Loading medium: 200 mM sodium acetate buffer, pH 3.5.

Base content prior to drug loading: 10.88 μmol per 80 μmol DPPC. 10.88μmol per loading.

Loading efficiency: Loading efficiency for 9.9 μmol buprenorphine was89.77% (Table 5, Example 4b). Surprisingly, this highly efficientloading occurred at pH 3.5, indicating the benefits of using2-methoxypyridine for buprenorphine loading. Buprenorphine has quitelimited solubility owing to its high hydrophobicity. Without being boundby theory, it was contemplated that the lower pKa of 2-methoxypyridine(3.28) would provide a lower intraliposomal pH prior to loading.Accordingly, a favorable pH gradient for drug loading exists even whenthe extraliposomal pH is only 3.5. Further, the residual2-methoxypyridine in the liposomes prior to drug loading was quite lowcompared to the amount that was observed for liposomes containing acomparable concentration of pyridinium sulfate. Without being bound bytheory, it is contemplated that a significant portion of the loss of2-methoxypyridinium sulfate is due to conversion of sulfate ions tobisulfate, which has a pKa of 1.98.

Leakage. The loss of buprenorphine from these liposomes followingloading was followed with the sedimentation method (FIG. 9, Example 4b).3.95% of the liposome contents were released in the first 48 hours,while by 552 hours the total loss was 7.94%, a release rate of 0.35% perday. This rate is much slower than the leakage rate observed forbuprenorphine loaded using pyridinium sulfate. The lower pKa of2-methoxypyridine (3.28, compared to 5.3 for pyridine) provides a lowerintraliposomal pH, which slows the release of buprenorphine. Theresidual 2-methoxypyridine may also be a more effective sacrificialrelease agent for buprenorphine than pyridine.

Example 4c: Buprenorphine Loaded Using 1 M 2-Methoxypyridinium Triflate

Loading base solution: 1 M 2-methoxypyridinium triflate (1 M withrespect to 2-methoxypyridine).

Loading medium: 200 mM sodium citrate buffer, pH 3.5.

Base content prior to drug loading: 48.6 μmol per 80 μmol DPPC. 48.6μmol per loading.

Loading efficiency: Loading efficiency for 9.9 μmol buprenorphine was86.3% (Table 5, Example 4c). Surprisingly, this high loading efficiencyoccurred at pH 3.5, indicating again the benefit of using2-methoxypyridine in preference to pyridine for buprenorphine loading,since it has quite limited solubility owing to its high hydrophobicity.The residual amount of 2-methoxypyridine in the liposomes was muchhigher than the residual amount of 2-methoxypyridinium sulfate observedin the previous experiments. Triflate has no pKa comparable tobisulfate, and therefore does not permit additional loss through theabsorption of protons by the acid ion.

Leakage. The loss of buprenorphine from these liposomes followingloading was followed using the sedimentation method (FIG. 9, Example4c). There is an initial release of 0.7% after 24 hours and a totalrelease of 3.45% of the liposome contents in the first 384 hours, arelease rate of 0.2% per day. While the initial release is lower, therate after 168 hours is very similar to that seen for buprenorphineloaded using 2-methoxypyridinium sulfate. Therefore, while the higherretention of 2-methoxypyridine in the liposomes using this salt reducesinitial leakage, the overall leakage rate is higher than the rateobserved for 2-methoxypyridinium sulfate. It is contemplated that thisreflects the lower long-term leakage with sulfate versus triflate.

Example 4d: Buprenorphine Loaded Using 1.6875 M Pyridazinium Triflate

Loading base solution: 1.6875 M pyridazinium triflate (1.6875 M withrespect to pyridazine).

Loading medium: 200 mM sodium citrate buffer, pH 3.5.

Base content prior to drug loading: 22.1 μmol per 80 μmol DPPC. 22.1μmol per loading.

Loading efficiency: Loading efficiency for 9.9 μmol buprenorphine was90.82% (Table 5, Example 4d). As with 2-methoxypyridine, this highefficiency loading occurred at pH 3.5, showing the benefit of using aloading base having a pKa that is comparable to the pKa of pyridazine(2.33). The residual amount of pyridazine in the liposomes was lowerthan that observed for 2-methoxypyridinium triflate. Triflate has no pKacomparable to bisulfate, and therefore would not permit additional lossthrough the absorption of protons by the acid ion. However, it iscontemplated that the lower pKa of pyridazine results in anintraliposomal pH low enough to protonate the phosphate of the DPPC.

Leakage. The loss of buprenorphine from these liposomes followingloading was followed using the sedimentation method (FIG. 9, Example4d). The release observed was very low: 1.04% leaked after 168 hours, arelease rate of 0.14% per day.

TABLE 5 Loading of buprenorphine buprenorphine buprenorphine addedcaptured Example (μmol) (%) 4a 17.86 44.47 4b 9.91 89.77 4c 9.91 86.314d 9.91 90.82

Example 5: Chloroquine Loading Using Adenine and Aniline

During the development of embodiments of the technology provided herein,experiments were conducted to test liposome loading with other weakbases, e.g., adenine and aniline The data collected indicated thatloading liposomes according to the technology provided is not limited tothe use of the weak base pyridine or its derivatives.

TABLE 6 Chloroquine loading with 0.5M adenine triflate lipid compositionDPPC:cholesterol 2:1 loading base solution 0.5M adenine triflate (0.5Mwith respect to adenine, supersaturated). loading medium 220 mM trischloride buffer, pH 8.3 base content prior to drug 27.55 μmol per 80μmol DPPC loading μmol chloroquine added 24 percent of chloroquineloaded 74.76

Adenine is a weak base (pKa 4.15) that has very limited solubility inwater. Unlike pyridine, 2-methoxypyridine, and pyridazine, adenine is asolid at room temperature. The solubility of adenine is greatlyincreased when converted to a salt of a strong acid, e.g., triflic acid.In the experiments described herein, adenine triflate was supersaturatedby warming to 75° C. immediately prior to use.

TABLE 7 Chloroquine loading using 1.5M aniline triflate lipidcomposition DPPC:cholesterol 2:1 loading base solution 1.5M anilinetriflate (1.5M with respect to aniline) loading medium 220 mM trischloride buffer, pH 8.3 base content prior to drug 20.66 μmol per 80μmol DPPC loading μmol chloroquine added 24 percent of chloroquineloaded 54.36

Aniline is a weak base (pKa 4.19). Aniline is a hydrophobic liquid withlimited solubility in water. Aniline forms salts with strong acids suchas triflic acid. Experiments were conducted using the salt anilinetriflate to load chloroquine into liposomes and subsequently monitor theleakage of chloroquine from the liposomes following loading.

Data were collected comparing the leakage of chloroquine from liposomesloaded using adenine triflate or aniline triflate with leakage ofchloroquine from liposomes loaded using pyridinium sulfate (FIG. 10).For all three loading bases, the data indicated that leakage begins witha very small initial release of around 2% of the captured drug, risingto 6-9% over 350 hours (FIG. 10). Leakage is quite comparable for allthree loading bases, though leakage is most rapid for aniline and leastrapid for adenine. Therefore, the data indicate that these threedifferent weak bases having similar pKa values though quite differentphysical properties produce chloroquine loaded liposomes with similarleakage characteristics.

Example 6: Doxycycline Loading with 2-Methoxypyridinium Sulfate

Doxycycline is an antibacterial drug whose therapeutic use would greatlyimprove if incorporated into a controlled release formulation. Forexample, doxycycline has been loaded into liposomes using acid loadingtechniques (see, e.g., U.S. patent application Ser. No. 14/030,131,incorporated herein by reference). During the development of embodimentsof the technology provided herein, experiments were conducted toincorporate doxycycline into liposomes using 2-methoxypyridiniumsulfate. Data collected indicated that doxycycline loaded into liposomesusing a weak base had a remarkably slow release profile for the drug.

TABLE 8 Doxycycline loading using 2-methoxypyridinium sulfate lipidcomposition DPPC:cholesterol 2:1 loading base solution 1.0M2-methoxypyridinium sulfate (2M with respect to 2-methoxypyridine)loading medium 200 mM sodium citrate buffer, pH 3.5 base content priorto drug 17.09 μmol per 80 μmol DPPC loading μmol doxycycline added 16percent of doxycycline loaded 87.03

The initial leakage of doxycyline was low, e.g., approximately 2%, andthe subsequent release rate was extremely low, e.g., only 13.34%released in 2300 hours. See, e.g., FIG. 11. This is an improved (e.g.,lower) rate of release for doxycycline from liposomes relative topresent technologies (see, e.g., Franklin (2015), Drug Metab Dispos 43:1236-45).

Example 7: Buprenorphine Loading Using Acids

Buprenorphine and other drugs have been efficiently loaded intoliposomes using acid loading techniques (see, e.g., U.S. patentapplication Ser. No. 14/030,131, incorporated herein by reference). Forexample, methods comprise preparing liposomes in a solution of acid(e.g., sulfuric acid), adding the drug, and neutralizing the externalacid solution. In addition, experiments conducted during the developmentof embodiments of the technology provided herein indicated that otheracids, e.g., hydrochloric acid and eprodisic acid, provide efficientloading. Furthermore, the data collected from experiments usingeprodisic acid indicated that eprodisic acid provided an improved (e.g.,slow) early phase of leakage, similar to that achieved with salts ofweak bases.

TABLE 9 Buprenorphine loaded using 1M hydrochloric acid lipidcomposition DPPC:cholesterol 2:1 loading solution 1.0M hydrochloric acidloading medium 200 mM sodium acetate buffer, pH 3.5, adjusted with NaOHμmol buprenorphine added 9 percent of buprenorphine loaded 68.65

TABLE 10 Buprenorphine loaded using 0.5M eprodisic acid lipidcomposition DPPC:cholesterol 2:1 loading solution 0.5M eprodisic acidloading medium 200 mM sodium citrate buffer, pH 3.5, adjusted with NaOHbase content prior to drug 20.66 μmol per 80 μmol DPPC loading μmolbuprenorphine added 9 percent of buprenorphine loaded 73.39

Leakage of buprenorphine from liposomes following acid loading was rapidwhen the acid used for loading was hydrochloric acid. However, releasewas very limited when the loading acid was eprodisic acid (see, e.g.,FIG. 12). Hence, use of eprodisic acid provides an improved acid loadingtechnology. Accordingly, embodiments of the technology comprise use ofeprodisic acid and eprodisate salts to improve the releasecharacteristics of drugs loaded into liposomes.

Example 8: Chloroquine Loading Using Salts of Eprodisic Acid

During the development of embodiments of the technology provided herein,experiments were conducted to test the loading of chloroquine intoliposomes using salts of eprodisic acid. In particular, chloroquine wasloaded into liposomes using 0.5 M 2-methoxpyridinium eprodisane and 0.5M pyridinium eprodisate.

TABLE 11 Chloroquine loaded using 0.5M 2-methoxypyridinium eprodisatelipid composition DPPC:cholesterol 2:1 loading base solution 0.5M2-methoxypyridinium eprodisate. (1.0M with respect to 2-methoxypyridine)loading medium 200 mM sodium citrate buffer, pH 3.5 base content priorto drug 26.19 μmol per 80 μmol DPPC. loading μmol chloroquine added 24percent of chloroquine loaded 74.71

TABLE 12 Chloroquine loaded using 0.5M pyridinium eprodisate lipidcomposition DPPC:cholesterol 2:1 loading base solution 0.5M pyridiniumeprodisate. (1.0M with respect to pyridine) loading medium 220 mM trischloride buffer, pH 8.3 base content prior to drug loading 41.96 μmolper 80 μmol DPPC μmol chloroquine added 24 percent of chloroquine loaded85.71

Buprenorphine loaded with eprodisic acid is released more slowlycompared to buprenorphine loaded with hydrochloric acid or sulfuric acid(see, e.g., FIG. 12). The leakage of drugs loaded with a weak basesulfate salt or a weak base chloride salt is lower than is seen forsulfuric acid loaded drugs. Moreover, leakage is further reduced byusing an eprodisate salt for loading. For example, the data collectedduring the development of embodiments of the technology indicated slowerleakage for chloroquine loaded into liposomes with either2-methoxypyridinium eprodisate or pyridinium eprodisate relative to theleakage of chloroquine loaded into liposomes using 0.5 M pyridiniumsulfate or 0.5 M 2-methoxypyridinium sulfate (FIG. 13). While leakage ofchloroquine loaded with a sulfate salt of pyridine is 4-8% after 400hours, leakage of chloroquine loaded with the eprodisate salts of bothpyridine and 2-methoxypyridine is only 1% or less after 400 hours.Therefore, eprodisate salts provide an improved salt for loadingliposomes relative to sulfate or chloride salts for drug release.

Example 9: Loading Using 0.2 M Nicotinamide Sulfate

Nicotinamide is a weak base, pKa 3.25, which, structurally, is apyridine. Unlike pyridine, 2-methoxypyridine, and pyridazine, it is asolid at room temperature. The solubility of nicotinamide is greatlyincreased when converted to a salt of a strong acid, giving solutions asconcentrated as 4M at pH 2-3 and room temperature with most strongacids. Nicotinamide is a highly desirable choice for drug loading,because it is the amide of Vitamin B3, nicotinic acid. Therefore, thereare no issues associated with administration of materials that maycontain it to human or animal subjects. Nicotinamide may be administeredorally at doses as high as 3 gm/day with no toxic effects.

TABLE 13 Doxycycline loading using 0.2M Nicotinamide Sulfate LipidComposition DPPC:cholesterol 2:1 Loading Base Solution 0.2M nicotinamidesulfate (0.4M with respect to nicotinamide). Loading medium 200 mMsodium citrate buffer, pH 3.5. Base Content Prior to Drug 83.13 μmol per80 μmol Phospholipon Loading 90H μmol Doxycycline Added 74.82 Percent ofDoxycycline Loaded 69.5

The pKa of nicotinamide is comparable to that of 2-methoxypyridine,making it a suitable base to use for loading at the low pH required forworking with buprenorphine and doxycycline. Table 13 shows thatdoxycycline can be loaded efficiently at pH 3.5 using nicotinamidesulfate.

TABLE 14 Naltrexone Loading using 0.2M Nicotinamide Sulfate. LipidComposition DPPC:cholesterol 2:1 Loading Base Solution 0.2M nicotinamidesulfate (0.4M with respect to nicotinamide). Loading medium 220 mMsodium citrate buffer, pH 7.0. Base Content Prior to Drug 33.18 μmol per26 μmol Phospholipon Loading 90H μmol Naltrexone Added 29.86 Percent ofNaltrexone Loaded 86.28

Nicotinamide sulfate was also used to load naltrexone into liposomessuccessfully, as shown in table 14. Naltrexone loading may be done athigher pH values than loading of doxycycline, and this is reflected inthe higher efficiency of loading for naltrexone.

Example 10: In Vivo Administration of Buprenorphine in Liposomes

During the development of embodiments of the technology provided herein,buprenorphine was loaded into liposomes using sulfate or eprodisatesalts (see Tables 15-18) and administered to rats to measure serumconcentrations of buprenorphine as a function of time for the liposomepreparations. Serum buprenorphine concentrations were monitoredfollowing a single 2 mg/Kg injection of buprenorphine loaded inliposomes.

TABLE 15 Buprenorphine Loaded into DPPC liposomes using 0.24M2-methoxypyridinium sulfate lipid composition DPPC:cholesterol 2:1loading base solution 0.24M 2-methoxypyridinium sulfate. (0.48M withrespect to 2-methoxypyridine) loading medium 200 mM sodium citratebuffer, pH 3.5 base content prior to drug loading 6.13 μmol per 80 μmolDPPC μmol buprenorphine added  9 percent of buprenorphine loaded 49.78

TABLE 16 Buprenorphine loaded into DSPC liposomes using 0.24M2-methoxypyridinium sulfate lipid composition DSPC:cholesterol 2:1loading base solution 0.24M 2-methoxypyridinium sulfate. (0.48M withrespect to 2-methoxypyridine) loading medium 200 mM sodium citratebuffer, pH 3.5 base content prior to drug loading 14.43 μmol per 80 μmolDPPC μmol buprenorphine added  9 percent of buprenorphine loaded 83.41

TABLE 17 Buprenorphine loaded into DPPC liposomes using 0.24M2-methoxypyridinium eprodisate lipid composition DPPC:cholesterol 2:1loading base solution 0.24M 2-methoxypyridinium eprodisate. (0.48M withrespect to 2-methoxypyridine) loading medium 200 mM sodium citratebuffer, pH 3.5 base content prior to drug loading 6.28 μmol per 80 μmolDPPC μmol buprenorphine added  9 percent of buprenorphine loaded 67.58

TABLE 18 Buprenorphine loaded into DSPC liposomes using 0.24M2-methoxypyridinium eprodisate lipid composition DSPC:cholesterol 2:1loading base solution 0.24M 2-methoxypyridinium eprodisate. (0.48M withrespect to 2-methoxypyridine) loading medium 200 mM sodium citratebuffer, pH 3.5 base content prior to drug loading 23.76 μmol per 80 μmolDPPC μmol buprenorphine added  9 percent of buprenorphine loaded 81.93

Data were collected from the administration of buprenorphine loaded intoliposomes using sulfate salts. FIG. 14 shows the serum concentrations inrats following a single subcutaneous injection of 2 mg/Kg buprenorphinein liposomes. Two preparations were studied in this experiment, whichare summarized in Table 15 and Table 16.

The serum concentration of buprenorphine loaded and administered in DPPCliposomes was approximately 20 ng/mL in the first 48 hours afteradministration (FIG. 14). After one week, the concentration declined toapproximately 4 ng/mL and then plateaued at a value of 1 ng/mL at 2-4weeks (FIG. 14).

The serum concentration of buprenorphine loaded and administered in DSPCliposomes was lower during the first 24 hours (approximately 2-3 ng/mL)and peaked at approximately 10-20 ng/mL at 48 hours to 1 week afteradministration (FIG. 14). Thereafter, the serum concentration plateauedat approximately 1 ng/mL (FIG. 14). Generally, liposomes prepared fromDSPC are more stable than those prepared from DPPC and this is reflectedin the lower concentrations at 4-24 hours and the later peak serumconcentrations (FIG. 14).

Data were collected from the administration of buprenorphine loaded intoliposomes using eprodisate salts. FIG. 15 shows the serum concentrationsin rats following a single injection of 2 mg/Kg buprenorphine inliposomes. Two preparations were studied in this experiment, which aresummarized in Table 17 and Table 18.

The serum concentration of buprenorphine loaded and administered in DPPCliposomes was approximately 6 ng/mL in the first 48 hours. After oneweek, the concentration declined to approximately 4 ng/mL and thenplateaued at a value of 0.6 ng/mL at 2-4 weeks.

The serum concentration of buprenorphine loaded and administered in DSPCliposomes was lower during the first 24 hours (e.g., approximately 2-3ng/mL) and peaked at approximately 6 ng/mL at 1 week. Thereafter,plateau concentrations of approximately 1 ng/mL were detected.

Accordingly, these data indicate that the peak serum concentrations ofbuprenorphine administered in liposomes loaded with 2-methoxypyridiniumeprodisate (which occur during the first week) are approximately 40% ofthe peak serum concentrations of buprenorphine detected whenbuprenorphine is administered in liposomes loaded with2-methoxypyridinium sulfate salt. This is consistent with the observedlower release rate seen for eprodisate-loaded drugs. The differencebetween DPPC and DSPC is very similar for the two salts. The dataindicated a long-term maintenance of therapeutically significantbuprenorphine serum concentrations in rats, paralleling the low in vitrorelease rates of buprenorphine from liposomes prepared and loadedaccording to the technology provided herein. In conclusion, eprodisateappears to be beneficial in providing serum concentrations that are moreconstant over time following injection.

Example 11: Liposome Preparation by Solvent Dilution

In some embodiments, liposomes are prepared by suspending aphospholipid:cholesterol mixture in chloroform, removing the chloroform,resuspending the lipid in t-butanol, freeze-drying the solution, andthen suspending the lipid lyophilizate in a solution containing eitheran acid or a salt. However, most phospholipids and cholesterol areprovided by commercial suppliers as solids. Accordingly, provided hereinare embodiments of a method of liposome preparation that eliminatesseveral steps, including the use of chloroform, to provide a method withfewer steps.

For example, in some embodiments of the method, phospholipid is mixedwith an alcohol to provide a solution of phospholipid in the alcohol.For example, during experiments conducted during the development ofembodiments of the technology, 60 mg of a commercial phosphatidylcholine(PHOSPHOLIPON 90H, comprising at least 90% hydrogenatedphosphatidylcholine) and 15 mg of cholesterol were mixed in a tube.Then, 100 microliters of 2-propanol were added to the lipid/cholesterolmixture. The mixture was warmed to 53 to 58° C., which provided a clearsolution of lipids in the alcohol solvent.

Then, 0.25 mL (a first volume or an initial volume) of a weak base salt(e.g., pyridinium sulfate or 2-methoxypyridinium sulfate) warmed to thesame temperature as the lipid in alcohol (e.g., 53 to 58° C.) was addedto the solution of lipids in the alcohol warmed at 53 to 58° C. Afteradding the weak base salt, a viscous, turbid suspension of liposomes wasformed. The mixture was mixed gently for 5 minutes, after which anadditional 0.25 mL (a second volume) of the base salt was added withfurther mixing. The resultant suspension was cooled to room temperature,below the phase transition temperature of the phospholipid, and thendiluted with 0.9% w/v NaCl (saline) to 13 mL. The suspension wassedimented and washed to eliminate unincorporated base, and the basecontent of the liposomes was then measured spectroscopically accordingto procedures described above.

TABLE 19 Incorporation of base salts into liposomes using solventdilution base content (mol/mol) base hydration of solvent saltconcentration base salt lyophilized lipid dilution 0.25M  pyridiniumsulfate ND 0.94 0.5M 0.334 1.32 1.0M 0.756 2.54 1.5M 1.8  3.16 0.25M 2-methoxypyridinium 0.075 ± 0.002 0.35 sulfate 0.5M 0.158 ± 0.044 0.581.0M 0.885 ± 0.612 1.81 0.2M pyridinium eprodisate ND 0.93 0.2M2-methoxypyridinium ND 0.32 eprodisate 0.25M  0.079 0.49

Table 19 shows the amount of either pyridine or 2-methoxypyridineincorporated into liposomes using the solvent dilution technologydescribed herein (Table 19, “solvent dilution”). Data collected forincorporation of pyridine and 2-methoxypyridine into liposomes usingtraditional chloroform dissolution, drying, and hydration are given forcomparison (Table 19, “hydration of lyophilized lipid”). The datacollected indicate that the base content for liposomes prepared byhydration rises steeply as the base salt concentration is increased.Without being bound by theory, it is contemplated that this relationshipreflects an increase in the capture volume of the liposomes, possiblycaused by solvation effects of these two bases. In contrast, two-stepsolvent dilution as described herein provides base incorporation that isproportional to the salt concentration. Furthermore, base incorporationproduced by the technology described herein is more efficient than baseincorporation provided by hydration. Base capture was much greater for agiven base salt concentration, and the efficiency is effectively doubledby use of smaller base salt volumes (e.g., 1 mL was used to hydratelipids in the traditional method and 0.5 mL was used to prepareliposomes in the solvent dilution method). In addition, the solventdilution technology described herein is highly controllable and isamenable to scale-up, e.g., including but not limited to production ofliposomes comprising sulfate salts and eprodisate salts of both pyridineand 2-methoxypyridine.

Example 12: Volume Ratios for Alcohol Dilution

As described above, embodiments of the solvent dilution method involvethe two-step addition of an aqueous solution to a lipid mixture inalcohol. In the particular experimental preparations described above,the first and second volumes added were each 250 microliters. During thedevelopment of embodiments of the technology, experiments were conductedto test capture of weak base salt in liposomes as a function of the twovolumes of base salt added to the preparation. In particular, liposomeswere prepared by dissolving 60 mg of a commercial phosphatidylcholine(PHOSPHOLIPON 90H, comprising at least 90% hydrogenatedphosphatidylcholine) and 15 mg cholesterol in 100 microliters of2-propanol at 58° C. Then, a “first volume” of pyridinium sulfate or2-methoxypyridinium sulfate (2-MP) was added and the solution was warmedat 58° C. for 5 minutes (Table 20). Next, a “second volume” ofpyridinium sulfate or 2-methoxypyridinium sulfate was added (Table 20)and the mixture was cooled to room temperature and diluted in 0.99% w/vNaCl.

TABLE 20 Encapsulation efficiency of 0.2M pyridinium sulfate and 0.2M2-methoxypyridinium sulfate as a function of volume of base salt addedto lipid in 2-propanol First Pyridine 2-MP Volume Second PyridineCapture 2-MP Capture (micro- Volume Capture Efficiency CaptureEfficiency liter) (microliter) (mol/mol) (%) (mol/mol) (%) 150 350 0.53721.48 0.067 2.68 200 300 0.511 20.44 0.116 4.64 250 250 0.718 28.720.265 10.6 300 200 ND 0.258 10.32 350 150 1.057 42.28 0.433 17.32 400100 1.136 45.44 0.185 7.4 450 50 1.012 40.48 ND — 500 0 0.860 34.4 ND —Data were collected during the experiments from measurements of theencapsulation efficiency of 0.2 M pyridinium sulfate or 0.2 M2-methoxypyridinium sulfate as a function of the initial volume of basesalt added (Table 20). The data indicated that the most efficientcapture of 2-methoxypyridine occurs when the lipid suspended in 100microliters of 2-propanol is diluted first with 350 microliters ofloading base, held at 58° C. for 5 minutes, and then diluted with afurther 150 microliter of loading base solution before cooling anddiluting with saline (Table 20). For pyridinium salts, the dataindicated that the highest capture occurs by adding 400 microliters ofloading base, incubating at 58° C. for 5 minutes, then adding 100microliters of loading base before cooling and diluting with saline(Table 20). These data indicate that the method provides a highefficiency of capture. For example, the best observed loading ofpyridine by this method was an incorporation efficiency of approximately45% of the added pyridinium salt being incorporated into the liposomes.Efficiency of capture of 2-methoxypyridine is lower.

Example 13: Choice of Alcohol for Alcohol Dilution Method

During the development of embodiments of the technology describedherein, similar experiments were conducted to measure the capture of 0.2M pyridinium and 2-methoxypyridinium sulfate in liposomes usingt-butanol (Table 21). As before, the lipid was suspended in 100microliters of the alcohol (t-butanol) at 62° C. and the first volume ofbase salt was added. After 5 minutes at 62° C., the second volume ofbase salt was added, the mixture was cooled and washed with salinebefore measuring the incorporated base content.

TABLE 21 Encapsulation efficiency of 0.2M pyridinium sulfate and 0.2M2-methoxypyridinium sulfate as a function of the volume of base saltadded to lipid in t-butanol. First Pyridine 2-MP Volume Second PyridineCapture 2-MP Capture (micro- Volume Capture Efficiency CaptureEfficiency liter) (microliter) (mol/mol) (%) (mol/mol) (%) 200 300 ND —0.118 4.72 250 250 0.87 34.8 ND — 300 200 ND — 0.147 5.88 400 100 ND —0.207 8.28 500 0 0.93 37.2 0.290 11.6 600 0 0.95 31.66 0.373 12.43 700 00.84 24.0 0.239 6.83 800 0 1.97 49.25 ND — 1000 0 0.13 2.6 ND — 1200 00.20 3.33 ND — 1400 0 0.17 2.43 ND —

The data collected indicated that a higher initial volume of base saltis required for efficient loading using lipids dissolved in t-butanol(Table 21) than the volume of base salt required for efficient loadingusing lipids dissolved in 2-propanol (Table 20). Despite this, theefficiency of loading of pyridinium sulfate with t-butanol issignificantly higher than for 2-propanol at the ratios providing thehighest loading efficiencies. Loading efficiency, however, is lower witht-butanol than with 2-propanol for 2-methoxypyridinium sulfate. Duringthe development of the embodiments described herein, experiments werealso conducted to compare the efficacy of t-butanol and 2-propanol toethanol and methanol. In particular, liposomes were prepared bydissolving 60 mg of a commercial phosphatidylcholine (PHOSPHOLIPON 90H,comprising at least 90% hydrogenated phosphatidylcholine) and 15 mgcholesterol in 100 microliters of each alcohol tested at the preparationtemperature indicated (Table 22).

TABLE 22 Capture efficiency of 0.2M pyridinium sulfate or 0.2M2-methoxypyridinium sulfate as a function of alcohol number preparationpyridine capture 2-MP capture of temperature (mol/mol (mol/mol Alcoholcarbons (° C.) phospholipid) phospholipid) Methanol 1 59 0.0595 0.007Ethanol 2 60 0.238 0.0015 2-propanol 3 59 0.718 0.283 t-butanol 4 620.869 0.161Then, 250 microliters of prewarmed 0.2 M pyridinium sulfate or2-methoxypyridinium sulfate (2-MP) were added to the dissolved lipid inalcohol and the mixture was incubated at the preparation temperature for5 minutes. Next, an additional 250 microliters of prewarmed 0.2 Mpyridinium sulfate or 2-methoxypyridinium sulfate was then added and themixture was cooled to room temperature. The liposomes were then dilutedwith 0.99% w/v NaCl and non-encapsulated pyridine was removed by threesedimentations in a bench top centrifuge at 1600×g for 5 minutes.Pyridine content in the resuspended final pellet was measuredspectroscopically. The data collected during the experiments indicatedthat 2-propanol and t-butanol provided incorporation of weak base intoliposomes by the solvent dilution method that was better thanincorporation of weak base into liposomes by the solvent dilution usingmethanol or ethanol (Table 22).

In addition, experiments were conducted during the development ofembodiments of the technology to test the use of 1-propanol (Table 23),2-butanol (Table 24), and 1-butanol (Table 25). In particular, Table 23shows the loading of 2-methoxypyridinium sulfate and pyridinium sulfateusing 1-propanol as the alcohol for lipid dissolution, Table 24 showsthe loading of 2-methoxypyridinium sulfate and pyridinium sulfate using2-butanol as the alcohol for lipid dissolution, and Table 25 shows theloading of pyridinium sulfate using 1-butanol.

The data collected during the experiments indicated that 1-propanol(Table 23) was more effective in some embodiments of the technology than2-propanol and t-butanol (compare Table 23 with Tables 20 and 21) withrespect to the amount and the percentage of pyridine incorporated intothe liposomes. In addition, the data indicated that high loadingefficiencies were provided by preparing liposomes by adding 500-600microliters of the aqueous phase to the lipid solution in the alcoholfor pyridinium sulfate and adding 700 microliters of the aqueous phaseto the lipid solution in the alcohol for 2-methoxypyridinium sulfate(Table 23). These volumes are greater for 1-propanol than for2-propanol. Given that 1-propanol has a log P of 0.329 and 2-propanolhas a log P of 0.16, some embodiments of the methods using 1-propanolmay require more dilution to achieve the desired alcohol concentrationin the lipid for optimal liposome formation and solute capture.

TABLE 23 Capture of pyridinium and 2-methoxypyridinium salts by alcoholdilution using 1-propanol First Pyridine 2-MP Volume Second PyridineCapture 2-MP Capture (micro- Volume Capture Efficiency CaptureEfficiency liter) (microliter) (mol/mol) (%) (mol/mol) (%) 100 400 1.7168.48 ND — 200 300 1.41 56.45 0.21 7.64 300 200 1.16 46.25 0.17 6.33 400100 1.05 41.87 0.17 6.24 500 0 1.72 68.93 0.39 13.76 600 0 1.93 64.450.47 13.65 700 0 1.59 45.51 0.96 24.11 800 0 ND — 0.56 12.33Although 2-butanol is not miscible with water in all ratios, it hassignificant solubility in aqueous solutions. In particular, 2-butanolhas an aqueous solubility of 290 g/L and a density of 0.808 g/mL.Accordingly, 2-butanol mixes freely with aqueous solutions when anaqueous volume of at least 0.2-0.3 mL is added to 0.1 mL 2-butanol. Inthe case of pyridinium salts, 2-butanol (Table 24) provides higherloading efficiencies than 2-propanol (Table 18), both in terms of theamount and the percentage of pyridine incorporated into the liposomes.Further, 2-butanol provides higher loading than 1-propanol (Table 23) interms of the amount of salt captured, though not in terms of maximumcapture efficiency. For the volumes that produce the most efficientloading, the volume of aqueous phase added to the lipid solution in thealcohol is greater for 2-butanol than it is for either 1-propanol or2-propanol. Given that 2-butanol has a log P of 0.683, 1-propanol has alog P of 0.329, and 2-propanol has a log P of 0.16, some embodiments ofthe methods using 2-butanol may require more dilution to achieve thedesired alcohol concentration in the lipid for optimal liposomeformation and solute capture.

TABLE 24 Capture of pyridinium and 2-methoxypyridinium salts by alcoholdilution using 2-butanol First Pyridine 2-MP Volume Second PyridineCapture 2-MP Capture (micro- Volume Capture Efficiency CaptureEfficiency liter) (microliter) (mol/mol) (%) (mol/mol) (%) 200 400 0.4317.35 ND 300 300 0.49 19.52 ND 400 200 0.52 20.62 ND 500 100 0.39 15.58ND 600 0 0.94 31.39 ND 700 0 1.44 41.19 ND 800 0 2.26 56.60 0.37 9.31900 0 2.69 59.68 ND 1000 0 3.06 61.24 0.54 10.77 1100 0 2.94 53.41 ND1200 0 2.62 43.70 0.34 5.641-butanol is also not fully miscible with water, and has a solubility of72 g/L. An optimization study for the capture of 0.2 M pyridiniumsulfate in liposomes using 1-butanol is shown in table 25. As before,the lipid was suspended in 100 microliter of the alcohol, t-butanol, at60° C., and the base salt was added. After 5 minutes at 60° C., themixture was then cooled and washed with saline before measuring the basecontent. 1-butanol requires addition of greater volumes of base salt forliposome formation. This is believed to be required because 1-butanol isa more hydrophobic alcohol with less water solubility than either2-butanol or 1-propanol. Efficiency of encapsulation appears to be lessthan is achieved with 1-propanol or 2-butanol, yet this alcohol mightstill find use in certain circumstances.

TABLE 25 Encapsulation efficiency of 0.2M pyridinium sulfate as afunction of the volume of base salt added to lipid in 1-butanol.Pyridine Pyridine Volume Capture Capture (microliter) (mol/mol)Efficiency (%) 600 0.748 24.94 800 0.914 22.86 1000 1.057 21.14 12001.613 26.88 1400 1.457 20.82 1600 2.093 26.47 1800 1.565 17.39 20001.629 16.29

Methanol, ethanol, 1-propanol, 2-propanol, and t-butanol are fivealcohols that are miscible with aqueous solutions; and, while 2-butanol,1-butanol, and isobutanol are not fully miscible in aqueous solutions,these alcohols have significant aqueous solubility. Accordingly, theefficacy of 2-butanol and 1-butanol (above) for the preparation ofliposomes indicates that the technology encompasses the use of alcoholswith solubility of at least 50 g/liter, preferably 150 g/liter (e.g.,alcohols with 1-6 carbon atoms, preferably 2-4 carbon atoms) in variousembodiments of the technology to provide efficient liposome loading.

Example 14: Capture of Other Solutes Using Solvent Dilution

During the development of embodiments of the technology, experimentswere conducted to measure the capture of the drugs doxycycline andchloroquine into liposomes using the solvent dilution methods describedherein. The data collected indicated that the solvent (e.g., alcohol)dilution method is generally applicable, e.g., to a wide range ofsolutes and not limited to salts of hydrophobic liquids.

Doxycycline hyclate was dissolved in water at 8.25 mg/mL and capturedusing 2-propanol dilution. In particular, 60 mg of a commercialphosphatidylcholine (PHOSPHOLIPON 90H, comprising at least 90%hydrogenated phosphatidylcholine) and 15 mg cholesterol were dissolvedin 100 microliters of 2-propanol at 58° C. Then, the initial volume ofthe doxycycline solution (pre-warmed to 58° C.) was added to thedissolved lipid (Table 23). After a 5-minute incubation at 58° C., thesecond volume of the doxycycline solution (pre-warmed to 58° C.) wasadded and the mixture was cooled to room temperature, washed withsaline, and the incorporation efficiency was measured (Table 26).

TABLE 26 Doxycycline direct capture using 2-propanol dilution doxycylinedoxycycline initial second capture capture volume volume efficiency(mol/mol (microliter) (microliter) (%) phospholipid) 200 300 33.1080.033 250 250 26.322 0.026 300 200 30.527 0.031 350 150 25.845 0.026

Efficient capture for this drug appears less dependent on the initialvolume ratio. The highest efficiency of incorporation achieved was about33% and occurred when the initial volume of doxycycline solution was 200microliters.

Chloroquine diphosphate was dissolved in 150 mM citrate buffer (pH 4.0)at a concentration of 200 mM and captured using 1-propanol dilution.Using the methods described above, 60 mg Phospholipon 90H and 15 mgcholesterol were dissolved in 100 microliters 1-propanol at 58° C. Then,the first volume of chloroquine solution (prewarmed to 58° C.) was addedto the dissolved lipid solution. The mixture was incubated for 5 minutesat 58° C.; then, the second volume of chloroquine solution (prewarmed to58° C.) was added and the mixture was cooled to room temperature. Thecapture results are shown in table 27.

TABLE 27 Chloroquine direct capture using 1-propanol dilutionchloroquine chloroquine capture capture initial volume second volumeefficiency (mol/mol (microliter) (microliter) (%) phospholipid) 300 20029.82 0.373 400 100 39.70 0.496 500 0 56.45 0.706 600 0 48.79 0.610

The data indicated that the most efficient capture of chloroquine wasapproximately 56% at an initial volume of 500 μl (Table 27). This is ahigher efficiency than doxycycline capture using 2-propanol, which isconsistent with the superior efficiency of 1-propanol for capture ofpyridine and 2-methoxypyridine discussed herein.

Example 15: Control of Leakage Rate by Selection of Counterion

Data collected during the development of embodiments of the technologyprovided herein indicate that the use of weak base loading inconjunction with sulfate or eprodisate produces liposome preparationshaving extremely low release (leakage) rates. However, a minimizedrelease rate may not be desirable in all circumstances. Accordingly,some embodiments provide a technology to tailor the release rate asappropriate for the desired application.

During the development of embodiments of the technology, experimentswere conducted to test control of release as a function of the weak basesalt used for loading. In particular, liposomes were prepared from acommercial phosphatidylcholine (PHOSPHOLIPON 90H, comprising at least90% hydrogenated phosphatidylcholine) and cholesterol at a 2:1 molarratio using 2-propanol dilution. In the experiments, pyridinium salts ofsulfate, eprodisate, methanesulfonate, benzenesulfonate, chloride, andnitrate were compared (Table 28). All pyridinium salts used were 0.4 Mwith respect to pyridine; hence the sulfate and eprodisate salts were0.2 M with respect to sulfate or eprodisate and 0.4 M with respect tothe other salts.

A mass of 60 mg of phosphatidylcholine and 15 mg cholesterol were mixedwith 100 microliters of 2-propanol at 58° C. to form a clear suspension.Then, 400 microliters of a pyridinium salt solution (prewarmed to 58°C.) were added to the lipid suspension. The mixture was kept at 58° C.for 5 minutes and then 100 microliters of the pyridinium salt solutionwere added. The mixture was cooled and diluted with 0.99% w/v sodiumchloride. After dilution, unencapsulated pyridine was removed bysedimentation in a benchtop centrifuge at 300×g for 5 minutes. A totalof three washes were performed. Naltrexone solution was then added in150 mM citrate (pH 7.0) and the mixture was left to load for 48 hours.After loading, residual non-loaded naltrexone and released pyridine wereremoved by dilution with saline and sedimentation a total of threetimes. Naltrexone and pyridine content were measured spectroscopically.The samples were subsequently monitored for release of naltrexone usingthe sedimentation method. The results of loading for the six differentsalts are summarized in Table 28. Two trials were performed for eachcondition.

TABLE 28 Naltrexone loading using pyridinium salts pyridinium saltpyridine content naltrexone percent before loading added naltrexone(micromol) (mg) loaded Experiment 1 2 1 2 1 2 Sulfate 58.19 64.02 19.7721.75 86.1 88.5 Eprodisate 74.67 74.83 25.37 25.43 85.6 84.2Methanesulfonate 60.97 74.26 20.81 25.34 89.0 89.3 Benzenesulfonate73.76 65.39 25.17 22.32 87.92 89.45 Chloride 63.06 64.91 21.52 22.1588.24 89.74 Nitrate 43.90 55.78 14.98 19.02 78.6 75.0

The data collected indicated that all pyridinium salts produced loadingof naltrexone in excess of 75%, though nitrate was somewhat lessefficient than the other salts, which all produced loading in excess of85%.

In further experiments, data were collected with respect to the releaseof naltrexone from liposomes loaded with different pyridinium salts(FIG. 16). Loading with pyridinium sulfate and pyridinium eprodisatesalts produced liposomes having a very low release rate, with only 5% orso release in 900 hours (FIG. 16). This is comparable to resultsobtained with buprenorphine and doxycycline. Benzenesulfonate andmethanesulfonate salts produce liposomes having higher release rates.Without being bound by theory, this observation was expected becausethese are monobasic acids. The chloride salt gave a more rapid 20%release in about 300 hours; nitrate salt release was the fastest withover 70% release in 500 hours and 90% in 860 hours (FIG. 16). Release inall cases was uniform, with no evidence of an initial fast rate ofrelease. These data indicate that the technology provided hereinprovides liposome formulations that release drug at a preciselydetermined and selected rate. Accordingly, the technology finds use inspecific applications associated with a need to control the life time ofa controlled release formulation.

All publications and patents mentioned in the above specification areherein incorporated by reference in their entirety for all purposes.Various modifications and variations of the described compositions,methods, and uses of the technology will be apparent to those skilled inthe art without departing from the scope and spirit of the technology asdescribed. Although the technology has been described in connection withspecific exemplary embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled in the artare intended to be within the scope of the following claims.

We claim:
 1. A composition comprising: liposomes, a bioactive agent, anda loading base selected from the group consisting of a pyridine, apyridine derivative, an adenine, an adenine derivative, an aniline, andan aniline derivative, wherein the liposomes have an intraliposomalspace and the liposome composition has an extraliposomal space, whereinthe intraliposomal space comprises the loading base in its charged andprotonated form, wherein the loading base is a weaker base than thebioactive agent, and an extraliposomal space comprising a loading mediumhaving a pH that is greater than the pKa of the charged and protonatedform of the loading base, wherein the bioactive agent is different fromthe loading base.
 2. The liposome composition of claim 1 wherein theloading medium further comprises a buffer.
 3. The liposome compositionof claim 1 wherein the concentration of the loading base in theintraliposomal space is greater than the concentration of the loadingbase in the extraliposomal space.
 4. The composition according to claim1 wherein the pKa of the charged and protonated form of the loading baseis less than 6, less than 5, less than 4, less than 3, or less than 2.5. The composition of claim 1 wherein the liposomes comprisephosphatidylcholine.
 6. The composition of claim 1 wherein the liposomescomprise: a) a phosphatidylcholine selected from the group consisting ofdistearoylphosphatidylcholine, hydrogenated soy phosphatidylcholine,hydrogenated egg phosphatidylcholine, dipalmitoylphosphatidylcholine,and dimyristoylphosphatidylcholine; b) a sphingomyelin; c) a neutrallipid; or d) an acidic phospholipid.
 7. The composition of claim 1wherein the liposomes comprise dipalmitoylphosphatidylcholine andoptionally cholesterol.
 8. The composition of claim 1, wherein theloading base is pyridine.